Methods and compositions for measuring beta cell death

ABSTRACT

Provided herein are, inter alia, nucleic acids, methods, and kits for detecting unmethylated DNA in body fluid sample of a subject. The disclosure includes compositions, methods, and kits for detecting unmethylation at a CpG site in an insulin gene promoter of a subject.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/144,816, filed Apr. 8, 2015, which is hereby incorporated in itsentirety and for all purposes.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file 48440-567001US_ST25.TXT, created onApr. 7, 2016, 7,485 bytes, machine format IBM-PC, MS-Windows operatingsystem, is hereby incorporated by reference.

BACKGROUND OF THE DISCLOSURE

Beta cell dysfunction and death is, in part, due to autoimmunity againstbeta cells, which contributes to the rising blood glucose level inpatients with type 1 diabetes (T1D). There is a correlation betweenautoantibody positivity and loss of beta cell function. However, inorder to identify individuals not only at risk but with actual ongoingdisease prior to loss of metabolic control, a direct measure of betacell death is needed. Provided herein are solutions to these and otherproblems in the art.

BRIEF SUMMARY OF THE DISCLOSURE

The current disclosure provides, inter alia, a quantitative methylationspecific nested primer based polymerase chain reaction (qMSP) assay, toidentify individuals not only at risk of T1D, but with actual ongoingdisease prior to loss of metabolic control. The compositions, methods,and kits provided in this disclosure allow, for example, functionalscreening of at risk individuals, monitoring of ongoing disease, as wellas in evaluating treatment methods including islet cell transplantation.The subject matter provided herein further relates to, inter alia,compositions, methods, and kits detecting unmethylated deoxyribonucleicacid (DNA) in blood of a subject. The disclosure further provides, interalia, compositions, methods, and kits for detecting unmethylation ofisolated DNA from blood of a subject at a CpG site in an insulin genepromoter located at −19 bp, −69 bp, −135 bp, −206 bp, or −357 bprelative to the transcription start site. In embodiments the insulingene promoter is on DNA from beta cells and isolated from blood of asubject.

The present disclosure provides a method of detecting unmethylatedpromoter DNA in insulin gene. The method involves modifying DNA isolatedfrom whole blood, plasma, serum, or a tissue sample of a subject toproduce a modified DNA. In the method of the present disclosure, DNA iscontacted with bisulfite for modifying unmethylated cytosine at a CpGsite in the DNA to uridine. The DNA is modified by deamination ofcytidine (or nucleoside cytosine) to uridine (or nucleoside uracil). Themodified DNA is amplified by using quantitative methylation-specificpolymerase chain reaction (qMSP) with methylation specific primers. Themethod provides detecting unmethylation at a CpG site.

In another aspect, the present disclosure provides nucleotides of SEQ IDNO: 1, 2, 3, and 4. The disclosure further provides nucleic acidcomprising SEQ ID NO: 1, 2, 3, or 4, hybridized to a complementary DNAsequence, where the complementary DNA sequence is modified to haveuridine.

In a further aspect, kits are provided, in which a first nucleic acidand a second nucleic acid each independently including sequences of SEQID NO: 1, 2, 3, 4, 5, or 6, are included. In the kit, the first andsecond nucleic acids do not simultaneously include the same sequence ofSEQ ID NO: 1, 2, 3, 4, 5, or 6. In another aspect, the presentdisclosure provides a kit including nucleic acids of SEQ ID NO: 1, 2, 3,4, 5, and 6.

In another aspect, the disclosure provides detecting unmethylatedinsulin gene promoter in blood of a subject as a measure of beta celldeath and a prognostic indicator of autoimmunity resulting in type 1diabetes (T1D).

In another aspect, the disclosure provides a method for treating anautoimmunity against insulin producing beta cells in a subject havingunmethylated CpG at −19 bp, −69 bp, −135 bp, −206 bp, and/or −357 bprelative to the transcription start site of an insulin gene promoter,determined by the method as set forth in this disclosure. Inembodiments, the insulin gene promoter is that of DNA from beta cells.

In another aspect, the disclosure provides a system including: at leastone processor; and at least one memory including program code which whenexecuted by the one memory provides operations that includes collectingDNA data associated with a subject; contacting said isolated DNA withbisulfite for modifying unmethylated cytosine at a CpG site in said DNAto uridine detecting unmethylation of DNA at said CpG site in an insulingene promoter located at −19 bp, −69 bp, −135 bp, −206 bp, or −357 bprelative to the transcription start site; and providing, via a userinterface, a prognosis and/or diagnosis for the subject based at leastin part on detected unmethylated DNA.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

Unless noted to the contrary, all publications, references, patentsand/or patent applications reference herein are hereby incorporated byreference in their entirety for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show detection of circulating beta cell DNA in non-obesediabetic (NOD) mice using qMSP assay. FIG. 1A depicts bar graphs ofblood glucose levels in NOD mice. FIG. 1B depicts a histogram of thedegree of insulitis, and FIG. 1C depicts bar graphs of circulatingunmethylated beta cell-specific DNA levels. Fold changes inunmethylation were quantified by calculation of the RelativeUnmethylation Ration (RUR) for each sample. The data display themean±standard error mean (SEM) of three independent measurements. Thestatistical significance was calculated by unpaired t tests comparedwith week 8 values and indicated by asterisks (*, P<0.05; **, P<0.01:***P<0.001, ****P<0.0001).

FIG. 2 depicts a histogram of tissue-specific methylation of the humaninsulin gene (INS) promoter. Statistics were done using the QUMAcomputer program (Kumaki et al., (2008), Nucl. Acids Res., 36 (suppl.2): W170-W175) and Fisher exact test comparing each site with the samesite in beta cells. The statistical significance is indicated byasterisks (*, P<0.1; **, P<0.01).

FIG. 3 depicts histogram of tissue methylation pattern of the human INSexon 2 (8CpG), intron 1 (2 CpG) and intron 2 (2 CpG). Statistics weredone using the QUMA computer program and Fisher exact test comparingeach site with the same site in beta cells. The statistical significanceis indicated by asterisks (*, P<0.1; **, P<0.01).

FIGS. 4A-4E show primer selection and analytical performance ofmethylation-specific PCR. FIG. 4A depicts a schematic of the human INSgene promoter region, showing the position of nine CpG sites and primerdesign for quantitative methylation-specific PCR assays. FIG. 4B showsan agarose gel electrophoresis of MSP reactions showing the size of thePCR products. FIG. 4C depicts a graph of real-time PCR data showinglinearity of Cq versus log copy number of unmethylated plasmid usingP20/P21 primer combination. FIG. 4D depicts a graph of real-time PCRdata showing linearity of Cq versus log copy number of unmethylatedplasmid using P40/P41 primer combination. FIG. 4E depicts a graph ofreal-time PCR data showing linearity of Cq versus log copy number ofunmethylated plasmid using P38/P39 primer combination.

FIGS. 5A-5B show histograms of beta cell specificity of methylationspecific primer (MSP) using bisulfate-converted genomic DNA (gDNA)obtained from human islets, blood, and colon used as a template fornested PCR using either bisulfite specific primers (BSP) (FIG. 5A) orMSP (FIG. 5B). The data display the mean±SEM of the RelativeUnmethylation Ratio (RUR). The cloned INS promoter was used fornormalization and standardization of the results. Statisticallysignificant differences between islets and other tissues were calculatedusing two-way ANOVA and the significance level indicated by asterisks(P<0.0001).

FIGS. 6A-6B show histograms of quantitative MSP for monitoring betacells in islet transplant patients. FIG. 6A depicts bar graph resultsusing blood samples, and FIG. 6B depicts bar graph results using plasmasamples. The data display the mean±SEM of the Relative UnmethylationRatio (RUR) calculations. The statistical significance was calculatedwith the Wilcoxon test to compare RUR of samples after transplant withthat before transplant and significance level indicated by asterisks (*,P<0.05; **, P<0.01).

FIGS. 7A-7AA show histograms of MSP assay and Mixed Meal Tolerance Test(MMTT) of patients. FIGS. 7A-7C show MSP and MMTT of patient 1; FIGS.7D-7F show MSP and MMTT of patient 2; FIGS. 7G-7I show MSP and MMTT ofpatient 3; FIGS. 7J-7L show MSP and MMTT of patient 4; FIGS. 7M-7O showMSP and MMTT of patient 5; FIGS. 7P-7R show MSP and MMTT of patient 6;FIGS. 7S-7U show MSP and MMTT of patient 7; FIGS. 7V-7X show MSP andMMTT of patient 8; and FIGS. 7Y-7AA show MSP and MMTT of patient 9.

FIG. 8 shows a system diagram illustrating a system for detectingunmethylated DNA in a subject, in accordance with some exampleembodiments.

FIG. 9 shows a flowchart illustrating a process for detectingunmethylated DNA in a subject, in accordance with some exampleembodiments.

DETAILED DESCRIPTION OF THE DISCLOSURE

Provided herein are, inter alia, compositions, methods, and kits fordetecting unmethylated DNA. In some aspects, the present disclosureincludes compositions, methods, and kits for detecting unmethylated DNAof insulin gene from beta cells.

The following definitions are included for the purpose of understandingthe present subject matter and for constructing the appended patentclaims. Abbreviations used herein have their conventional meaning withinthe chemical and biological arts.

Definitions

The term “disease” refers to any deviation from the normal health of amammal and includes a state when disease symptoms are present, as wellas conditions in which a deviation (e.g., infection, gene mutation,genetic defect, etc.) has occurred, but symptoms are not yet manifested.According to the present disclosure, the methods disclosed herein aresuitable for use in a patient that is a member of the Vertebrate class,Mammalia, including, without limitation, primates, livestock anddomestic pets (e.g., a companion animal). Typically, a patient will be ahuman patient.

The term “Type 1 Diabetes (T1D)” is used in the usual customary sense,which is a condition in which the immune system destroysinsulin-producing cells of the pancreas, thereby compromising orreducing the ability to use glucose (blood sugar) for energy. T1Dusually occurs in children and young adults.

The terms “subject,” “patient,” “individual,” and the like as usedherein are not intended to be limiting and can be generallyinterchanged. That is, an individual described as a “patient” does notnecessarily have a given disease, but may be merely seeking medicaladvice.

The term “subject” as used herein includes all members of the animalkingdom prone to suffering from the indicated disorder. In some aspects,the subject is a mammal, and in some aspects, the subject is a human.

It must be noted that as used herein and in the appended embodiments,the singular forms “a,” “an,” and “the” include the plural referenceunless the context clearly dictates otherwise. Thus, for example, areference to “a disease,” “a disease state”, “a nucleic acid” or “a CpGsite” is a reference to one or more such embodiments, and includesequivalents thereof known to those skilled in the art and so forth.

Throughout the description and claims of this specification the word“comprise” and other forms of the word, such as “comprising” and“comprises,” means including but not limited to, and is not intended toexclude, for example, other components.

A “control” sample or value refers to a sample that serves as areference, usually a known reference, for comparison to a test sample.For example, a test sample can be taken from a patient suspected or atrisk of having T1D and compared to samples from a known T1D patient, ora known normal (non-disease) individual. A control can also represent anaverage value gathered from a population of similar individuals, e.g.,T1D patients or healthy individuals with a similar medical background,same age, weight, etc. A control value can also be obtained from thesame individual, e.g., from an earlier-obtained sample, prior todisease, or prior to treatment. One of skill will recognize thatcontrols can be designed for assessment of any number of parameters.

One of skill in the art will understand which controls are valuable in agiven situation and be able to analyze data based on comparisons tocontrol values. Controls are also valuable for determining thesignificance of data. For example, if values for a given parameter arewidely variant in controls, variation in test samples will not beconsidered as significant.

The term “diagnosis” refers to a relative probability that a disease ispresent in the subject. Similarly, the term “prognosis” refers to arelative probability that a certain future outcome may occur in thesubject. For example, in the context of the present disclosure,prognosis can refer to the likelihood that an individual will develop adisease, or the likely severity of the disease (e.g., severity ofsymptoms, rate of functional decline, survival, etc.). The terms are notintended to be absolute, as will be appreciated by any one of skill inthe field of medical diagnostics.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammaticalequivalents used herein means at least two nucleotides covalently linkedtogether. Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10,12, 15, 25, 30, 40, 50 or more nucleotides in length, up to about 100nucleotides in length. Nucleic acids, including ribonucleic acids (RNA)and deoxyribonucleic acids (DNA), and polynucleotides are a polymers ofany length, including longer lengths, e.g., 200, 300, 500, 1000, 2000,3000, 5000, 7000, 10,000, etc. A nucleic acid of the present inventionwill generally contain phosphodiester bonds, although in some cases,nucleic acid analogs are included that may have alternate backbones,comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate,or O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press); and peptidenucleic acid backbones and linkages. Other analog nucleic acids includethose with positive backbones; non-ionic backbones, and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Sanghui & Cook, eds. Nucleic acidscontaining one or more carbocyclic sugars are also included within onedefinition of nucleic acids. Modifications of the ribose-phosphatebackbone may be done for a variety of reasons, e.g., to increase thestability and half-life of such molecules in physiological environmentsor as probes on a biochip. Mixtures of naturally occurring nucleic acidsand analogs can be made; alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made.

The term “bp” and the like refer, in the usual and customary sense, tothe indicated number of base pairs.

The term “promoter” and the like in the usual and customary sense, is aregion of DNA that initiates transcription of a particular gene.Promoters are located near the transcription start sites of genes, onthe same strand and upstream on the DNA (towards the 5′ region of thesense strand). Upstream and downstream in the usual and customary senseboth refer to a relative position in DNA or RNA. Each strand of DNA orRNA has a 5′ end and a 3′ end, so named for the carbon position on thedeoxyribose (or ribose) ring. By convention, upstream and downstreamrelate to the 5′ to 3′ direction in which RNA transcription takes place.Upstream is toward the 5′ end of the RNA molecule and downstream istoward the 3′ end. When considering double-stranded DNA, upstream istoward the 5′ end of the coding strand for the gene in question anddownstream is toward the 3′ end. Due to the anti-parallel nature of DNA,this means the 3′ end of the template strand is upstream of the gene andthe 5′ end is downstream.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids (e.g., genomic sequences or subsequences or codingsequences) or polypeptide sequences, refer to two or more sequences orsubsequences that are the same or have a specified percentage of aminoacid residues or nucleotides that are the same (i.e., 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more identity over a specified region), when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection. Suchsequences are then said to be “substantially identical.” This definitionalso refers to the compliment of a test sequence. Optionally, theidentity exists over a region that is at least about 10 to about 100,about 20 to about 75, about 30 to about 50 amino acids or nucleotides inlength.

An example of algorithms suitable for determining percent sequenceidentity and sequence similarity are the BLAST and BLAST 2.0 algorithms,which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402(1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990),respectively. As will be appreciated by one of skill in the art, thesoftware for performing BLAST analyses is publicly available through thewebsite of the National Center for Biotechnology Information.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers, those containing modified residues, and non-naturallyoccurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs may have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins or otherentities which can be made detectable, e.g., by incorporating aradiolabel into a peptide or antibody specifically reactive with atarget peptide. Any method known in the art for conjugating an antibodyto the label may be employed, e.g., using methods described inHermanson, Bioconjugate Techniques 1996, Academic Press, Inc., SanDiego.

In some examples of the disclosed methods, when the expression level ofa biomarker(s) is assessed, the level is compared with controlexpression level of the biomarker(s). By control level is meant theexpression level of a particular biomarker(s) from a sample or subjectlacking a disease (e.g., T1D), at a selected stage of a disease ordisease state, or in the absence of a particular variable such as atherapeutic agent. Alternatively, the control level includes a knownamount of biomarker. Such a known amount correlates with an averagelevel of subjects lacking a disease, at a selected stage of a disease ordisease state, or in the absence of a particular variable such as atherapeutic agent. A control level also includes the expression level ofone or more biomarkers from one or more selected samples or subjects asdescribed herein. For example, a control level includes an assessment ofthe expression level of one or more biomarkers in a sample from asubject that does not have a disease (e.g., T1D), is at a selected stageof progression of a disease (e.g., T1D), or has not received treatmentfor a disease. Another exemplary control level includes an assessment ofthe expression level of one or more biomarkers in samples taken frommultiple subjects that do not have a disease, are at a selected stage ofprogression of a disease, or have not received treatment for a disease.

When the control level includes the expression level of one or morebiomarkers in a sample or subject in the absence of a therapeutic agent,the control sample or subject is optionally the same sample or subjectto be tested before or after treatment with a therapeutic agent or is aselected sample or subject in the absence of the therapeutic agent.Alternatively, a control level is an average expression level calculatedfrom a number of subjects without a particular disease. A control levelalso includes a known control level or value known in the art.

The term “cell death” includes “apoptosis”, “autophagy”, “necrosis”,“cornification”, “mitotic catastrophe”, “anoikis”, “excitotoxicity”,“wallerian degeneration”, “paraptosis”, “pyroptosis”, “pyronecrosis”,and “entosis”. A detailed review of certain cell death mechanisms isprovided, for example, in Kroemer et al., Classification of Cell Death,Cell Death Differ., (2009), 16(1): 3-11.

The term “programmed cell death” and the like refer, in the usual andcustomary sense, to death of a cell mediated by an intracellular programand carried out in a regulated process which typically confers advantageduring the life cycle of the organism. The term “apoptosis” and the likerefer, in the usual and customary sense, to a process of programmed celldeath characterized by biochemical events that lead to orderly cellchanges and eventual cell death. The term “autography” and the likerefer, in the usual and customary sense, to the natural destructivemechanism that disassembles unnecessary or dysfunctional cellularcomponents through a regulated process. Accordingly, apoptosis andautophagy are forms of programmed cell death. The term “non-programmedcell death” and the like refer, in the usual and customary sense, tocell death which is not programmed cell death, e.g., necrosis due toexternal factors such as trauma, infection, and the like.

The term “associated” or “associated with” in the context of a substance(e.g., unmethylated DNA of insulin gene promoter) or substance activity(e.g., unmethylated DNA of insulin gene promoter activity) or substancefunction (e.g., unmethylated DNA of insulin gene promoter function)associated with a disease does not necessarily mean that the disease iscaused by (in whole or in part), or a symptom of the disease is causedby (in whole or in part) the substance or substance activity or function(i.e., unmethylated DNA of insulin gene promoter, unmethylated DNA ofinsulin gene promoter activity, unmethylated DNA of insulin genepromoter function).

The term “unmethylated DNA” or “demethylated DNA” means DNA thatpartially or completely lacks a methyl group conjugated to cytosine in asegment of the DNA. In embodiments, an unmethylated DNA or demehtylatedDNA partially or completely lacks a methyl group conjugated to cytosinerelative to a control DNA. The control DNA may be, for example, may be aDNA derived from a healthy patient or patient population (e.g. a patientthat does not have Type I diabetes). Thus, in embodiments, anunmethylated DNA or demehtylated DNA partially or completely lacks amethyl group conjugated to cytosine relative to the methylation patternobserved on an equivalent DNA sequence derived from a patient that doesnot have Type I diabetes. DNA methylation typically occurs in a CpGdinucleotide context. In the context of the present disclosure, the DNAcan be equivalent to a short (2-50 nucleotides, e.g. 5-50 nucleotides)double stranded or single stranded nucleic acid, a nucleic acid fragmentcloned in a plasmid DNA, a nucleic acid fragment amplified from a sampleof a subject, and/or synthetically prepared a nucleic acid fragment. DNAmethylation at the 5′ position of cytosine may have the specific effectof reducing gene expression in vivo. DNA methylation may also form thebasis of chromatin structure, which typically enables a single cell togrow into multiple organs or perform multiple functions.

The CpG sites or CG sites are regions of DNA where a cytosine nucleotideoccurs next to a guanine nucleotide in the linear sequence of basesalong its length. “CpG” is shorthand for “-C-phosphate-G-”, that is,cytosine and guanine separated by only one phosphate; phosphate linksany two nucleosides together in DNA. The “CpG” notation is used todistinguish this linear sequence from the CG base-pairing of cytosineand guanine. The CpG notation can also be interpreted as the cytosinebeing 5′ prime to the guanine base.

In embodiments, Methylation-Specific PCR is based on a chemical reactionof sodium bisulfite with DNA that converts unmethylated cytosines of CpGdinucleotides to uracil or UpG, followed by traditional PCR. However,methylated cytosine is not converted in this process, and primers aredesigned to overlap the CpG site of interest, which allows one todetermine methylation status as methylated or unmethylated.

Relative Unmethylation Ratio (RUR) as used in this disclosure is basedon Relative Expression Ratio (RER) described by Husseiny et al. (2012),PloS One 7: e47942.

As used herein, the term “administering” means oral administration,administration as a suppository, topical contact, intravenous,parenteral, intraperitoneal, intramuscular, intralesional, intrathecal,intranasal or subcutaneous administration, or the implantation of aslow-release device, e.g., a mini-osmotic pump, to a subject.Administration is by any route, including parenteral and transmucosal(e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, ortransdermal). Parenteral administration includes, e.g., intravenous,intramuscular, intra-arteriole, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial. Other modes ofdelivery include, but are not limited to, the use of liposomalformulations, intravenous infusion, transdermal patches, etc.

The terms “administration” or “administering” refer to the act ofproviding an agent of the current embodiments or pharmaceuticalcomposition including an agent of the current embodiments to theindividual in need of treatment.

By “co-administer” it is meant that a composition described herein isadministered at the same time, just prior to, or just after theadministration of additional therapies. The compound or the compositionof the disclosure can be administered alone or can be co-administered tothe patient. Co-administration is meant to include simultaneous orsequential administration of the compound individually or in combination(more than one compound or agent). The preparations can also becombined, when desired, with other active substances (e.g. to reducemetabolic degradation).

As used herein, “sequential administration” includes that theadministration of two agents (e.g., the compounds or compositionsdescribed herein) occurs separately on the same day or do not occur on asame day (e.g., occurs on consecutive days).

As used herein, “concurrent administration” includes overlapping induration at least in part. For example, when two agents (e.g., any ofthe agents or class of agents described herein that has bioactivity) areadministered concurrently, their administration occurs within a certaindesired time. The agents' administration may begin and end on the sameday. The administration of one agent can also precede the administrationof a second agent by day(s) as long as both agents are taken on the sameday at least once. Similarly, the administration of one agent can extendbeyond the administration of a second agent as long as both agents aretaken on the same day at least once. The bioactive agents/agents do nothave to be taken at the same time each day to include concurrentadministration.

As used herein, “intermittent administration includes the administrationof an agent for a period of time (which can be considered a “firstperiod of administration”), followed by a time during which the agent isnot taken or is taken at a lower maintenance dose (which can beconsidered “off-period”) followed by a period during which the agent isadministered again (which can be considered a “second period ofadministration”). Generally, during the second phase of administration,the dosage level of the agent will match that administered during thefirst period of administration but can be increased or decreased asmedically necessary.

The compositions disclosed herein can be delivered transdermally, by atopical route, formulated as applicator sticks, solutions, suspensions,emulsions, gels, creams, ointments, pastes, jellies, paints, powders,and aerosols. Oral preparations include tablets, pills, powder, dragees,capsules, liquids, lozenges, cachets, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. Solid formpreparations include powders, tablets, pills, capsules, cachets,suppositories, and dispersible granules. Liquid form preparationsinclude solutions, suspensions, and emulsions, for example, water orwater/propylene glycol solutions. The compositions of the presentdisclosure may additionally include components to provide sustainedrelease and/or comfort. Such components include high molecular weight,anionic mucomimetic polymers, gelling polysaccharides and finely-divideddrug carrier substrates. These components are discussed in greaterdetail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760.The entire contents of these patents are incorporated herein byreference in their entirety for all purposes. The compositions disclosedherein can also be delivered as microspheres for slow release in thebody. For example, microspheres can be administered via intradermalinjection of drug-containing microspheres, which slowly releasesubcutaneously (see Rao, J. Bioniater Sci. Polym. Ed. 7:623-645, 1995;as biodegradable and injectable gel formulations (see, e.g., Gao Phann.Res. 12:857-863, 1995); or, as microspheres for oral administration(see, e.g., Eyles, J. Phann. Pharmacol. 49:669-674, 1997).

As used herein, an “effective amount” or “therapeutically effectiveamount” is that amount sufficient to affect a desired biological effect,such as beneficial results, including clinical results. As such, an“effective amount” depends upon the context in which it is beingapplied. An effective amount may vary according to factors known in theart, such as the disease state, age, sex, and weight of the individualbeing treated. Several divided doses may be administered daily or thedose may be proportionally reduced as indicated by the exigencies of thetherapeutic situation. In addition, the compositions/formulations ofthis disclosure can be administered as frequently as necessary toachieve a therapeutic amount.

As used herein, the term “cytokine” is a term used for proteins releasedby one cell population which act on another cell as intercellularmediators. Certain examples of such cytokines are lymphokines,monokines, and traditional polypeptide hormones. Included among thecytokines are, e.g., growth hormone such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepaticgrowth factor; fibroblast growth factor; prolactin; placental lactogen;tumor necrosis factor α and β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, β, and γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-la, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-16,IL-17, IL-18, IL-22, IL-23, IL-27, IL-35, IL-35; a tumor necrosis factorsuch as TNF-α or TNF-β; and other polypeptide factors including LIF andkit ligand (KL). As used herein, the term cytokine includes proteinsfrom natural sources or from recombinant cell culture and biologicallyactive equivalents of the native sequence cytokines.

Pharmaceutical compositions may include compositions wherein thetherapeutic drug (e.g., agents described herein, including embodimentsor examples) is contained in a therapeutically effective amount, i.e.,in an amount effective to achieve its intended purpose. The actualamount effective for a particular application will depend, inter alia,on the condition being treated. When administered in methods to treat adisease, such compositions will contain an amount of therapeutic drugeffective to achieve the desired result, e.g., modulating the activityof a target molecule, and/or reducing, eliminating, or slowing theprogression of disease symptoms.

The dosage and frequency (single or multiple doses) administered to amammal can vary depending upon a variety of factors, for example,whether the mammal suffers from another disease, and its route ofadministration; size, age, sex, health, body weight, body mass index,and diet of the recipient; nature and extent of symptoms of the diseasebeing treated, kind of concurrent treatment, complications from thedisease being treated or other health-related problems. Othertherapeutic regimens or agents can be used in conjunction with themethods and agents of this disclosure. Adjustment and manipulation ofestablished dosages (e.g., frequency and duration) are well within theability of those skilled in the art.

For any therapeutic agent described herein, the therapeuticallyeffective amount can be initially determined from cell culture assays.Target concentrations will be those concentrations of therapeuticdrug(s) that are capable of achieving the methods described herein, asmeasured using the methods described herein or known in the art.

As is well known in the art, therapeutically effective amounts for usein humans can also be determined from animal models. For example, a dosefor humans can be formulated to achieve a concentration that has beenfound to be effective in animals. The dosage in humans can be adjustedby monitoring agent's effectiveness and adjusting the dosage upwards ordownwards, as described above. Adjusting the dose to achieve maximalefficacy in humans based on the methods described above and othermethods is well within the capabilities of the ordinarily skilledartisan.

Dosages may be varied depending upon the requirements of the patient andthe therapeutic drug being employed. The dose administered to a patientshould be sufficient to effect a beneficial therapeutic response in thepatient over time. The size of the dose also will be determined by theexistence, nature, and extent of any adverse side-effects. Determinationof the proper dosage for a particular situation is within the skill ofthe practitioner. Generally, treatment is initiated with smaller dosageswhich are less than the optimum dose of the agent. Thereafter, thedosage is increased by small increments until the optimum effect undercircumstances is reached. Dosage amounts and intervals can be adjustedindividually to provide levels of the administered agent effective forthe particular clinical indication being treated. This will provide atherapeutic regimen that is commensurate with the severity of theindividual's disease state.

A weight percent of a component, unless specifically stated to thecontrary, is based on the total weight of the formulation or compositionin which the component is included.

The terms “treat,” “treating” or “treatment,” and other grammaticalequivalents as used herein, include alleviating, abating, ameliorating,or preventing a disease, condition or symptoms, preventing additionalsymptoms, ameliorating or preventing the underlying metabolic causes ofsymptoms, inhibiting the disease or condition, e.g., arresting thedevelopment of the disease or condition, relieving the disease orcondition, causing regression of the disease or condition, relieving acondition caused by the disease or condition, or stopping the symptomsof the disease or condition, and are intended to include prophylaxis.The terms further include achieving a therapeutic benefit and/or aprophylactic benefit. By therapeutic benefit is meant eradication oramelioration of the underlying disorder being treated. Also, atherapeutic benefit is achieved with the eradication or amelioration ofone or more of the physiological symptoms associated with the underlyingdisorder such that an improvement is observed in the patient,notwithstanding that the patient may still be afflicted with theunderlying disorder.

The terms “prevent,” “preventing,” or “prevention,” and othergrammatical equivalents as used herein, include to keep from developing,occur, hinder or avert a disease or condition symptoms as well as todecrease the occurrence of symptoms. The prevention may be complete(i.e., no detectable symptoms) or partial, so that fewer symptoms areobserved than would likely occur absent treatment. The terms furtherinclude a prophylactic benefit. For a disease or condition to beprevented, the compositions may be administered to a patient at risk ofdeveloping a particular disease, or to a patient reporting one or moreof the physiological symptoms of a disease, even though a diagnosis ofthis disease may not have been made.

The term “inhibiting” also means reducing an effect (disease state orexpression level of a gene/protein/mRNA) relative to the state in theabsence of a compound or composition of the present disclosure.

The terms “phenotype” and “phenotypic” as used herein refer to anorganism's observable characteristics such as onset or progression ofdisease symptoms, biochemical properties, or physiological properties.

The word “expression” or “expressed” as used herein in reference to aDNA nucleic acid sequence (e.g. a gene) means the transcriptional and/ortranslational product of that sequence. The level of expression of a DNAmolecule in a cell may be determined on the basis of either the amountof corresponding mRNA that is present within the cell or the amount ofprotein encoded by that DNA produced by the cell (Sambrook et al., 1989Molecular Cloning: A Laboratory Manual, 18.7-18.88). When used inreference to polypeptides, expression includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion. Expression can bedetected using conventional techniques for detecting protein (e.g.,ELISA, Western blotting, flow cytometry, immunofluorescence,immunohistochemistry, etc.).

The term “gene” means the segment of DNA involved in producing aprotein; it includes regions preceding and following the coding region(leader and trailer) as well as intervening sequences (introns) betweenindividual coding segments (exons). The leader, the trailer as well asthe introns include regulatory elements that are necessary during thetranscription and the translation of a gene. Further, a “protein geneproduct” is a protein expressed from a particular gene.

The term “an amount of” in reference to a polynucleotide or polypeptide,refers to an amount at which a component or element is detected. Theamount may be measured against a control, for example, wherein anincreased level of a particular polynucleotide or polypeptide inrelation to the control, demonstrates enrichment of the polynucleotideor polypeptide. Thus, in embodiments, an increased amount indicates agreater level or efficiency of grafting HSPCs described herein into ahost (e.g. mouse). The term refers to quantitative measurement of theenrichment as well as qualitative measurement of an increase or decreaserelative to a control.

Throughout the description and claims of this specification the word“comprise” and other forms of the word, such as “comprising” and“comprises,” means including but not limited to, and is not intended toexclude, for example, other components.

The term “about” refers to any minimal alteration in the concentrationor amount of an agent that does not change the efficacy of the agent inpreparation of a formulation and in treatment of a disease or disorder.The term “about” with respect to concentration range of the agents(e.g., therapeutic/active agents) of the current disclosure also refersto any variation of a stated amount or range which would be an effectiveamount or range.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it is understood thatthe particular value forms another aspect. It is further understood thatthe endpoints of each of the ranges are significant both in relation tothe other endpoint, and independently of the other endpoint. It is alsounderstood that there are a number of values disclosed herein, and thateach value is also herein disclosed as “about” that particular value inaddition to the value itself. It is also understood that throughout theapplication, data are provided in a number of different formats and thatthis data represent endpoints and starting points and ranges for anycombination of the data points. For example, if a particular data point“10” and a particular data point “15” are disclosed, it is understoodthat greater than, greater than or equal to, less than, less than orequal to, and equal to 10 and 15 are considered disclosed as well asbetween 10 and 15. It is also understood that each unit between twoparticular units are also disclosed. For example, if 10 and 15 aredisclosed, then 11, 12, 13, and 14 are also disclosed.

The term “beta cell” the like refer, in the usual and customary sense,to cells found in the pancreatic islets, the primary function of whichis storage and release of insulin.

Method of Detecting Unmethylated DNA

Provided herein is a method of detecting unmethylated DNA in a subject.The method includes detecting unmethylation of a DNA sample isolatedfrom a sample of a subject. In some aspects, the sample is a body fluidof a subject. The body fluid may be blood, saliva, and/or tears. In someaspects the blood is whole blood, plasma, or serum. In embodiments, theDNA sample is isolated from a tissue sample, e.g., stomach, spleen,lung, kidney, colon, pancreas, and/or breast, of the subject. The methodmay also include detecting unmethylation of a control DNA sample. Themethod may involve isolating DNA from a sample of the subject, anddetecting unmethylation of the isolated DNA at a CpG site. The methodprovides detecting unmethylation at CpG site in the insulin gene of asubject. For example, the method provides detecting unmethylation at aCpG site in the promoter region or an exon of the insulin gene. Forexample, the method provides detecting unmethylation at a CpG site inthe promoter region of the insulin gene. The method may also includedetecting unmethylation at a CpG site in an exon and an intron of theinsulin gene.

The present disclosure includes a method for detecting unmethylatedpromoter region of insulin gene, in which insulin DNA is treated withbisulfite to convert unmethylated cytosine to uracil while sparing anymethylated cytosine. The amplification of the bisulfite treated DNA withbisulfite-specific primer (BSP) using polymerase chain reaction (PCR)specifically amplifies methylated and unmethylated DNA. The amplifiedDNA using the BSP primers is then used to perform a second PCR, e.g.,methylation sensitive PCR, using two or more different methylationspecific primers (MSP) which amplifies a segment containing unmethylatedCpG site. The methylation specific primers of the present disclosurematch and hybridize to a complementary unmethylated template DNA. In thesecond-step of the PCR, the products from the first reaction are used asa template for quantitative PCR (qPCR) with nested primers. Inembodiments, the present disclosure includes a quantitative MSP (qMSP)method. In embodiments, qMSP of the present disclosure is sensitive andspecific for detection of rare DNA. The qMSP may also differentiatebetween methylated and unmethylated DNA by using oligonucleotide primerswhose 3′-ends match the methylation status of specific CpG sites in abisulfite treated template. In embodiments, two MSP PCRs are performedsequentially with two sets of MSPs, in which a first MSP PCR using oneset of nested methylation specific primers is followed by a second MSPPCR using a second set of nested methylation specific primers, whichdetects CpG sites at the insulin gene promoter. For example, the methodmay detect unmethylation at CpG site located at −19 bp, −69 bp, −135 bp,−206 bp, or −357 bp of the insulin gene (see Husseiny M I et al.,Tissue-specific methylation of human insulin gene and PCR assay formonitoring beta cell death, PLoS One. 2014 Apr. 10; 9(4):e94591), orsites equivalent thereto. In embodiments, the insulin gene is the humaninsulin gene. For example, the insulin gene is of pancreatic cells of ahuman subject. For example, the insulin gene is of the islets ofLangerhans of a human subject. For example, the insulin gene is of betacells (β cells) of the islets of Langerhans in the pancreas of a humansubject.

In embodiments, the beta cells of a human subject are undergoing celldeath. For example, non-limiting examples of cell death is due to“apoptosis”, “autophagy”, “necrosis”, “cornification”, “mitoticcatastrophe”, “anoikis”, “excitotoxicity”, “wallerian degeneration”,“paraptosis”, “pyroptosis”, “pyronecrosis”, and “entosis”. Inembodiments, the beta cells in a human subject are dying due to anautoimmune response. For example, an autoimmune response of a humansubject destroys or causes death of the beta cells.

The present disclosure includes a method of detecting unmethylated DNAin which DNA isolated from whole blood, plasma, serum, or a tissuesample of a subject is modified to produce a modified DNA. In the methodof the present disclosure, DNA is modified by deamination of cytidine(or nucleoside cytosine) to uridine (or nucleoside uracil). The modifiedDNA may include uridine. The modified DNA is amplified by usingquantitative methylation-specific polymerase chain reaction (qMSP) withmethylation specific primers. The unmethylation is detected at the CpGsite.

The method of the present disclosure may include obtaining a bloodsample from the subject. The DNA is isolated from the blood samples. TheDNA is modified with bisulfite. The DNA is amplified with bisulfitespecific primers. Quantitative methylation-specific polymerase chainreaction (qMSP) of the insulin gene promoter is performed withmethylation specific primers that bind to complementary sequences on theamplified bisulfite modified DNA. In embodiments, the unmethylation isdetected at a CpG site at −19 bp, −69 bp, −135 bp, −206 bp, and/or −357bp in the insulin gene promoter, or sites equivalent thereto. The bloodsample can be whole blood, plasma, or serum.

In embodiments, the insulin gene promoter is the underlined sequence endof SEQ ID NO: 13 or a fragment thereof, a nucleotide sequence of SEQ IDNO: 14, or a homolog thereof. In embodiments the CpG site at −19 bp, forexample, is a CG sequence with the “C” (i.e., cytosine) located 19 basepair upstream (i.e, 5′) of the transcription start site of SEQ ID NO: 13(italicized “AGC” sequence). In embodiments, the CpG site at −19 bp, forexample, would be an equivalent position upstream of the transcriptionstart site of a homologous insulin promoter. For example, the CpG sitesat −19 bp, −69 bp, −135 bp, −206 bp, and −357 bp at SEQ ID NO: 13 arebolded and italicized. In embodiments, CpG sites are located atequivalent positions on a homologous insulin gene promoter.

Human INS Gene

[SEQ ID NO: 13] GGGGACAGGGGTGTGGGGACAGGGGTGTGGGGACAGGGGTGTGGGGACAGGGGTCTGGGGACAGGGGTGTGGGGACAGGGGTCCTGGGGACAGGGGTGTGGGGATAGGGGTGTGGGGACAGGGGTGTGGGGACAGGGGTGTGGGGACAGGGGT CTGGGGACAGCAG 

CAAAGAGCCC 

CCCTGCAGCCTCCAGCTCTCCTGGTCTAATGTGGAAAGTGGCCCAGGTGAGGGCTTTGCTCTCCTGGAGACATTTGCCCCCAGCTGTGAGCAGGGACAGGTCTGGCCAC 

GGCCCCTGGTTA AGACTCTAATGACC 

CTGGTCCTGAGGAAGAGGTGCTGA 

ACCAAGG AGATCTTCCCACAGACCCAGCACCAGGGAAATGGTC 

GAAATTGCAGCC TCAGCCCCCAGCCATCTGC 

ACCCCCCCACCCCAGGCCCTAATGGGCC  AGG 

GCAGGGGTTGAGAGGTAGGGGAGATGGGCTCTGAGACTATAAAGC CAG 

GGGGCCCAGCAGCCCTC AGCCCTCCAGGACAGGCTGCATCAGAAGAGGCCATCAAGCAGGTCTGTTCCAAGGGCCTTTGCGTCAGGTGGGCTCAGGATTCCAGGGTGGCTGGACCCCAGGCCCCAGCTCTGCAGCAGGGAGGACGTGGCTGGGCTCGTGAAGCATGTGGGGGTGAGCCCAGGGGCCCCAAGGCAGGGCACCTGGCCTTCAGCCTGCCTCAGCCCTGCCTGTCTCCCAGATCACTGTCCTTCTGCCATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCTGGGGACCTGACCCAGCCGCAGCCTTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTACCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCCGGGAGGCAGAGGACCTGCAGGGTGAGCCAACTGCCCATTGCTGCCCCTGGCCGCCCCCAGCCACCCCCTGCTCCTGGCGCTCCCACCCAGCATGGGCAGAAGGGGGCAGGAGGCTGCCACCCAGCAGGGGGTCAGGTGCACTTTTTTAAAAAGAAGTTCTCTTGGTCACGTCCTAAAAGTGACCAGCTCCCTGTGGCCCAGTCAGAATCTCAGCCTGAGGACGGTGTTGGCTTCGGCAGCCCCGAGATACATCAGAGGGTGGGCACGCTCCTCCCTCCACTCGCCCCTCAAACAAATGCCCCGCAGCCCATTTCTCCACCCTCATTTGATGACCGCAGATTCAAGTGTTTTGTTAAGTAAAGTCCTGGGTGACCTGGGGTCACAGGGTGCCCCACGCTGCCTGCCTCTGGGCGAACACCCCATCACGCCCGGAGGAGGGCGTGGCTGCCTGCCTGAGTGGGCCAGACCCCTGTCGCCAGGCCTCACGGCAGCTCCATAGTCAGGAGATGGGGAAGATGCTGGGGACAGGCCCTGGGGAGAAGTACTGGGATCACCTGTTCAGGCTCCCACTGTGACGCTGCCCCGGGGCGGGGGAAGGAGGTGGGACATGTGGGCGTTGGGGCCTGTAGGTCCACACCCAGTGTGGGTGACCCTCCCTCTAACCTGGGTCCAGCCCGGCTGGAGATGGGTGGGAGTGCGACCTAGGGCTGGCGGGCAGGCGGGCACTGTGTCTCCCTGACTGTGTCCTCCTGTGTCCCTCTGCCTCGCCGCTGTTCCGGAACCTGCTCTGCGCGGCACGTCCTGGCAGTGGGGCAGGTGGAGCTGGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTTGGCCCTGGAGGGGTCCCTGCAGAAGCGTGGCATTGTGGAACAATGCTGTACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAACTAGACGCAGCCCGCAGGCAGCCCCACACCCGCCGCCTCCTGCACCGAGAGAGATGGAATAAAGCCCTTGAACCAGCCCTGCT.

Human INS promoter: In embodiments, the insulin gene promoter is SEQ IDNO: 14 or a fragment thereof. In embodiments, the CpG sites at −19 bp,−69 bp, −135 bp, −206 bp, and −357 bp at SEQ ID NO: 14 are bolded anditalicized, which are positions upstream (i.e., 5′) of the transcriptionstart site (SEQ ID NO: 14 does not include the transcription startsite). In embodiments, CpG sites are located at equivalent positions ona homologous insulin gene promoter.

[SEQ ID NO: 14] GACAGGGGTGTGGGGATAGGGGTGTGGGGACAGGGGTGTGGGGACAGGGGTGTGGGGACAGGGGTCTGGGGACAGCAG 

CAAAGAGCCC 

CCCTGCAG CCTCCAGCTCTCCTGGTCTAATGTGGAAAGTGGCCCAGGTGAGGGCTTTGCTCTCCTGGAGACATTTGCCCCCAGCTGTGAGCAGGGACAGGTCTGGCCAC 

GGCCCCTGGTTAAGACTCTAATGACC 

CTGGTCCTGAGGAAGAGGTG CTGA 

ACCAAGGAGATCTTCCCACAGACCCAGCACCAGGGAAATGGTC 

GAAATTGCAGCCTCAGCCCCCAGCCATCTGC 

ACCCCCCCACCCCA GGCCCTAATGGGCCAGG 

GCAGGGGTTGAGAGGTAGGGGAGATGGGCTC TGAGACTATAAAGCCAG 

GGGGCCCAGCAGCCCTC.

Bisulfite converted DNA of Unmethylated CG of Human INS promoter: Inembodiments, the bisulfite modified DNA of unmethylated CG sequence ofthe human INS promoter includes “TG” sequences located at −19 bp, −69bp, −135 bp, −206 bp, and −357 bp at SEQ ID NO: 15 are bolded andunderlined, which are positions upstream (i.e., 5′) of the transcriptionstart site (SEQ ID NO: 15 does not include the transcription startsite). In embodiments, the bisulfite modified DNA of unmethylated INSpromoter with “TG” sequences located at −19 bp, −69 bp, −135 bp, −206bp, and −357 bp at SEQ ID NO: 15 or equivalents thereof in a homologueis from an INS promoter from beta cells. In embodiments, bisulfitemodified sites are located at equivalent positions on a homologousinsulin gene promoter.

[SEQ ID NO: 15] GTGGGGATAGGGGTGTGGGGATAGGGGTGTGGGGATAGGGGTGTGGGGATAGGGGTTTGGGGATAGTAG 

TAAAGAGTTT 

TTTTGTAGTTTTTAGTT TTTTTGGTTTAATGTGGAAAGTGGTTTAGGTGAGGGTTTTGTTTTTTTGGAGATATTTGTTTTTAGTTGTGAGTAGGGATAGGTTTGGTTAT 

GGTTTTT GGTTAAGATTTTAATGATT 

TTGGTTTTGAGGAAGAGGTGTTGA 

AT TAAGGAGATTTTTTTATAGATTTAGTATTAGGGAAATGGTT 

GAAATTG TAGTTTTAGTTTTTAGTTATTTGT 

ATTTTTTTATTTTAGGTTTTAATG GGTTAGG 

GTAGGGGTTGAGAGGTAGGGGAGATGGGTTTTGAGATTATA AAGTTAG 

GGGGTTTAGTAGTTTTT.

Bisulfite converted DNA of Methylated CG of Human INS promoter: Inembodiments, the bisulfite modified DNA of methylated CG sequence of thehuman INS promoter includes “CG” sequences located at −19 bp, −69 bp,−135 bp, −206 bp, and −357 bp at SEQ ID NO: 16 are bolded andunderlined, which are positions upstream (i.e., 5′) of the transcriptionstart site (SEQ ID NO: 16 does not include the transcription startsite). In embodiments, bisulfite modified sites are located atequivalent positions on a homologous insulin gene promoter. Inembodiments, the bisulfite modified DNA of methylated INS promoter with“CG” sequences located at −19 bp, −69 bp, −135 bp, −206 bp, and −357 bpat SEQ ID NO: 16 or equivalents thereof in a homologue is from an INSpromoter, which is either not from beta cells or a control promoter. Inembodiments, the control promoter is from a subject not at risk of orsuffering from autoimmunity against beta cells.

[SEQ ID NO: 16] GTGGGGATAGGGGTGTGGGGATAGGGGTGTGGGGATAGGGGTGTGGGGATAGGGGTTTGGGGATAGTAG 

TAAAGAGTTT 

TTTTGTAGTTTTTAGTT TTTTTGGTTTAATGTGGAAAGTGGTTTAGGTGAGGGTTTTGTTTTTTTGGAGATATTTGTTTTTAGTTGTGAGTAGGGATAGGTTTGGTTAT 

GGTTTTT GGTTAAGATTTTAATGATT 

TTGGTTTTGAGGAAGAGGTGTTGA 

AT TAAGGAGATTTTTTTATAGATTTAGTATTAGGGAAATGGTT 

GAAATTG TAGTTTTAGTTTTTAGTTATTTGT 

ATTTTTTTATTTTAGGTTTTAATG GGTTAGG 

GTAGGGGTTGAGAGGTAGGGGAGATGGGTTTTGAGATTATA AAGTTAG 

GGGGTTTAGTAGTTTTT.

The method of the present disclosure includes methylation specificprimers, which hybridize to a CpG site of the insulin gene. For example,the primer hybridizes to a CpG site in the promoter region of the humaninsulin gene. The method includes detecting methylation or unmethylationat a CpG site in the promoter region of the human insulin gene usingspecific primers. The method includes methylation specific primershaving a sequence of at least SEQ ID NO: 1, 2, 3, and 4. Each of themethylation specific primers of the method does not simultaneously havea sequence of at least SEQ ID NO: 1, 2, 3, and 4. In some aspects, themethod includes a methylation specific primer that has a sequence thatis at least 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%,80%-85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical or homologous to a nucleic acid having a sequence of at leastSEQ ID NO: 1, 2, 3, or 4.

In embodiments, the methylation specific primers used in the method ofthe present disclosure are:

P20: H-Pro-Bisulf-For1 (SEQ ID NO: 1)5′-ATAGGGGTGTGGGGATAGGGGTTTGGGGATAGTAGT-3′ P21: H-Pro-Bisulf-Rev1(SEQ ID NO: 2) 5′-AACCCATCTCCCCTACCTCTCAACCCCTACCA-3′ P38: H-Pro-BS-For4(SEQ ID NO: 3) 5′-TGGGTTTTTGGTTAAGATTTTAATGATTT-3′ P39: H-Pro-BS-Rev5(SEQ ID NO: 4) 5'-CAACAAATAACTAAAAACTAAAACTACAATTTCCA-3′

In embodiments, the bisulfite specific primers are:

P40: MSP-For1 (SEQ ID NO: 5) 5′-ATAGGGGTGTGGGGATAGGGGTTTGGGGATAGTA-3′P41: MSP-Rev1 (SEQ ID NO: 6) 5′-CAAAACCCATCTCCCCTACCTCTCAACCCCTAC-3′

The primers may detect differentially methylated sites (e.g.unmethylated sites) in the promoter region of the human insulin gene.The method includes nested PCR technique that interrogates methylationsites with both high specificity and sensitivity. In embodiments, theprimers of the present disclosure are designed to recognize onlyunmethylated DNA as found in beta cells. Primers P20 (SEQ ID NO: 1) andP21 (SEQ ID NO: 2) are methylation-specific primers (FIG. 4A, dashedarrows) targeting CpGs at −357 bp and −69 bp, respectively, and togetherproduce a product of 350 bp (FIG. 4B). Primers P38 (SEQ ID NO: 3) andP39 (SEQ ID NO: 4) target CpGs −206 bp and −135 bp, respectively, andproduce a product of 130 bp (FIG. 4B). Primers P40 (SEQ ID NO: 5) andP41 (SEQ ID NO: 6), target the regions just upstream and downstream ofP20 (SEQ ID NO: 1) and P21 (SEQ ID NO: 2), respectively, and do notalign with any CpG site (FIG. 4A, solid arrows). P40 (SEQ ID NO: 5) andP41 (SEQ ID NO: 6) are bisulfite-specific (BSP) and amplify a 350 bpproduct from both methylated and unmethylated DNA, and therefore providea measure of total amplifiable insulin gene promoter sequences (FIG.4B). The BSP amplified product is used for detecting specificunmethylation using nested PCR. In embodiments, two nested PCRs areperformed sequentially. For example, in some aspects, PCR with P20 (SEQID NO: 1)/P21 (SEQ ID NO: 2) is followed by PCR with P38 (SEQ ID NO:3)/P39 (SEQ ID NO: 4) is performed. In some aspects, the two rounds ofnested MSP PCRs using with P20 (SEQ ID NO: 1)/P21 (SEQ ID NO: 2) isfollowed by PCR with P38 (SEQ ID NO: 3)/P39 (SEQ ID NO: 4) detects CpGsites by detecting unmethylation at −357/−69 bp and −206/−135 bp,respectively.

In embodiments, the method may include serial dilutions of a clonedunmethylated insulin gene as a template to evaluate the disclosedprimers. In embodiments, each MSP and BSP primer set of the presentdisclosure has a dose dependent amplification ranging from 10⁶ copies toas few as 5 copies of the unmethylated sequences (FIG. 4B). The presentdisclosure includes quantitative analysis of the standard curves, whichshows that the BSP and qMSP assays are linear over a 10⁵-fold range oftemplate concentrations (FIG. 4C). Variation across the nested MSP curveranged from 2.83% to 6.58% (Table 1).

TABLE 1 Statistical variation of qMSP standard curve. Log Copy AverageNumber C_(q) ±SD % CV 1.7 28.22 0.80 2.83 2.2 25.79 0.92 3.57 2.7 24.290.75 3.09 3.1 23.01 0.79 3.44 3.6 21.35 0.68 3.21 4.1 20.07 0.77 3.864.6 17.89 .0.68  3.84 5.0 14.58 0.83 5.68 5.5 14.36 0.48 3.35 6.0 10.030.66 6.58 C_(q) is the average of n = 5; SD = standard deviation, % CV =percent coefficient of variation [(SD/C_(q) average) × 100].

In embodiments, the primers sets may be tested for specificity andsensitivity employing serially diluted bisulfite converted gDNA fromhuman islets, blood, and colon as templates for qMSP. Fold changes inunmethylation may be calculated by the Relative Unmethylation Ratio(RUR) for each sample in which the level of beta cell DNA (qMSP) isnormalized for total amplifiable sequences (qBSP). To assess the effectof targeting 2 versus 4 CpG sites, the nested reaction using P38 (SEQ IDNO: 3)/P39 (SEQ ID NO: 4) may be preceded by a first reaction usingeither BSP primers (P40 (SEQ ID NO: 5)/P41 (SEQ ID NO: 6)) to target atotal of 2 sites or MSP primers (P20 (SEQ ID NO: 1)/P21 (SEQ ID NO: 2))to target 4 sites. The assay interrogating 2 CpG sites was shown toexhibit a highly significant specificity for islets over blood and colon(FIG. 5A). In embodiments, the assay targeting 4 sites shows a greaterdifference in signal between islets and other tissues, indicatingincreased specificity of the assay when the number of CpG sitesinterrogated is increased in the assay (FIG. 5B).

TABLE 2 The amplification efficiency of qMSP and qBSP standard curves.PCR type (Primer set) Efficiency % ± SD Slope ± SD R² ± SD Nested MSP(P38/P39) 85.19 ± 1.37 −3.737 ± 0.05 0.979 ± 0.004 BSP (P40/P41) 73.44 ±7.81 −4.212 ± 0.32 0.989 ± 0.01  MSP = methylation-specific PCR, BSP =bisulfite-specific PCR, Slope = slope of the standard curve, R² = thesquare of the correlation coefficient of the standard curve.

In embodiments, quantitative analysis of standard curves of BSP and qMSPassays are performed. In embodiments, the standard curves are linearover about a 10⁵-fold range of template concentrations (FIG. 4C). Realtime PCR may be used to generate labeled PCR products, e.g., SYBR® GreenPCR products, to document linearity of Cq versus log copy number ofunmethylated plasmid, e.g., from 5 to 10⁶ copies. For nested PCR of thepresent disclosure, the two MSP assays may be applied sequentially,i.e., amplification with P20/P21 followed by P38/P39. For example,variation across the nested MSP curve of the present disclosure rangedfrom 2.83% to 6.58% (see Table 1). Furthermore, the standard curveparameters (see Table 2) of the PCR reactions included in the presentdisclosure are reproducible for both nested qMSP (e.g.,efficiency=85.19%±1.37 SD, slope=−3.737±0.05 SD, R²=0.979±0.004 SD; n=5experiments) and qBSP (e.g., efficiency=73.44%±7.81 SD,slope=−4.212±0.32 SD, R²=0.989±0.01 SD; n=5 experiments)

The method of the present disclosure may be used to detect unmethylationat two or more CpG sites on the insulin gene promoter. In embodiments,the method does not detect unmethylation at a CpG site in the insulingene promoter located at −102 bp, −180 bp, −234 bp, or −345 bp.

The method of the present disclosure may be used to detect beta celldeath by detecting unmethylation at a CpG site at −19 bp, −69 bp, −135bp, −206 bp, or −357 bp in an human insulin gene promoter, or sitesequivalent thereto. In embodiments, beta cell death is not detected bydetecting unmethylation at −102 bp, −180 bp, −234 bp, and −345 bp onhuman insulin gene promoter.

Compositions

Provided herein are compositions of nucleic acids for detectingunmethylated DNA in a subject. In embodiments, the present disclosureincludes one or more nucleic acids having a sequence of SEQ ID NO: 1, 2,3, or 4. In embodiments, each of the nucleic acids is different. Thus,in embodiments, each of the nucleic acids do not simultaneously have thesame sequence selected from SEQ ID NO: 1, 2, 3, and 4. In embodiments,the nucleic acid has a sequence that is at least 50%-55%, 55%-60%,60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more identical or homologous to a nucleicacid having a sequence of at least SEQ ID NO: 1, 2, 3, or 4.

In embodiments, the present disclosure includes a nucleic acidcomprising SEQ ID NO: 1, 2, 3, or 4, where the nucleic acid ishybridized to a complementary DNA sequence. In embodiments, the nucleicacid is hybridized to a complementary DNA sequence of the presentdisclosure. The nucleic acid may have a sequence that is at least50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical or homologousto SEQ ID NO: 1, 2, 3, or 4. The complementary sequence can havehomology of at least 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%,75%-80%, 80%-85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore to a sequence complementary to SEQ ID NO: 1, 2, 3, or 4. Thecomplementary DNA to which the nucleic acid of the present disclosure ishybridized may include a uridine. In embodiments, the present disclosuremay include a nucleic acid hybridized to complementary DNA comprising aCpG site.

In embodiments, the present disclosure may further include a DNAcomprising a uridine. In embodiments, the DNA may be hybridized to afirst nucleic acid and a second nucleic acid each independentlycomprising SEQ ID NO: 1, 2, 3, or 4. In embodiments, the first and thesecond nucleic acids are different. In other words, in embodiments, thefirst and the second nucleic acids do not simultaneously include thesame SEQ ID NO: 1, 2, 3, or 4.

In some aspects the first nucleic acid includes SEQ ID NO: 1 and thesecond nucleic acid includes SEQ ID NO: 2. In embodiments, the firstnucleic acid includes SEQ ID NO: 3 and the second nucleic acid includesSEQ ID NO: 4. The DNA may be derived from a biological sample. Forexample, in embodiments the biological sample is from a human. Forexample, the biological sample may be whole blood, plasma, or serum of ahuman.

Kits

Provided herein are kits for detecting unmethylated DNA in a body fluidof a subject. In embodiments, the kits include reagents, such as nucleicacids, for detecting unmethylated DNA from a body fluid, e.g., blood(e.g., whole blood, plasma, and/or serum) of a subject, e.g., human. Inembodiments, the present disclosure includes a kit including a firstnucleic acid and a second nucleic acid each independently including SEQID NO: 1, 2, 3, 4, 5, or 6, in which the first and the second nucleicacids do not simultaneously include the same SEQ ID NO: 1, 2, 3, 4, 5,or 6. In embodiments, the present disclosure includes a kit with anucleic acid that may have a sequence having at least 50%-55%, 55%-60%,60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more identity or homology to a nucleic acidhaving a sequence of SEQ ID NO: 1, 2, 3, or 4.

In embodiments the present disclosure includes a kit containing twonucleic acids, the first nucleic acid including SEQ ID NO: 1 and thesecond nucleic acid including SEQ ID NO: 2. In embodiments, the presentdisclosure includes a kit containing two nucleic acids, with a first anda second nucleic acid that may have a sequence having at least 50%-55%,55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity or homology to anucleic acid having a sequence of SEQ ID NOs:1 and 2, respectively. Inembodiments, a kit of the present disclosure includes two nucleic acids,the first nucleic acid including SEQ ID NO: 3 and a second nucleic acidincluding SEQ ID NO: 4. In embodiments, the present disclosure includesa kit containing two nucleic acids, with a first and a second nucleicacid that may have a sequence having at least 50%-55%, 55%-60%, 60%-65%,65%-70%, 70%-75%, 75%-80%, 80%-85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identity or homology to a nucleic acid having asequence of SEQ ID NOs:3 and 4, respectively. In embodiments, thepresent disclosure includes a kit containing two nucleic acids, thefirst nucleic acid including SEQ ID NO: 5 and the second nucleic acidincluding SEQ ID NO: 6. In embodiments, the present disclosure includesa kit containing two nucleic acids, with a first and a second nucleicacid that may have a sequence having at least 50%-55%, 55%-60%, 60%-65%,65%-70%, 70%-75%, 75%-80%, 80%-85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identity or homology to a nucleic acid having asequence of SEQ ID NOs: 5 and 6, respectively.

In embodiments the present disclosure includes a kit containing fournucleic acids, the first nucleic acid including SEQ ID NO: 1, the secondnucleic acid including SEQ ID NO: 2, the third nucleic acid includingSEQ ID NO:3, and the fourth nucleic acid including SEQ ID NO: 4. Inembodiments, the present disclosure includes a kit containing fournucleic acids, with a first, a second, a third, and a fourth nucleicacid that may have a sequence having at least 50%-55%, 55%-60%, 60%-65%,65%-70%, 70%-75%, 75%-80%, 80%-85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identity or homology to a nucleic acid having asequence of SEQ ID NOs:1, 2, 3, and 4, respectively.

In embodiments, the present disclosure includes a kit containing sixnucleic acids, the first nucleic acid including SEQ ID NO: 1, the secondnucleic acid including SEQ ID NO: 2, the third nucleic acid includingSEQ ID NO:3, the fourth nucleic acid including SEQ ID NO: 4, the fifthnucleic acid including SEQ ID NO: 5, and the sixth nucleic acidincluding SEQ ID NO: 6. In embodiments, the present disclosure includesa kit containing six nucleic acids, with a first, a second, a third, afourth, a fifth, and a sixth nucleic acid that may have a sequencehaving at least 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%,80%-85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentity or homology to a nucleic acid having a sequence of SEQ IDNOs:1, 2, 3, 4, 5, and 6, respectively.

In embodiments of the present disclosure, the kit(s) may further includeenzymes, reagents for deamination of cytosine, buffers, vials, plasmidvectors, control DNA, devices for collecting blood and/or tissuesamples, or reagents for labeling DNA, or any combinations thereof. Theenzymes are, for example, thermostable DNA polymerase enzymes,restriction enzymes, and combination thereof.

In embodiments, the present disclosure includes a kit including reagentsfor detecting beta cell death in a subject. The reagents are, e.g., forobtaining blood sample from the subject; isolating DNA from the bloodsample; modifying the DNA with bisulfite; amplifying the modified DNAwith bisulfite specific primers; performing quantitativemethylation-specific polymerase chain reaction (qMSP) of a gene segment,e.g., insulin gene promoter, with methylation specific primers that bindto complementary sequences on the amplified bisulfite modified DNA; anddetecting unmethylation at a CpG site at −19 bp, −69 bp, −135 bp, −206bp, and −357 bp in the insulin gene promoter, or equivalents thereof.

In embodiments, the kit of the present disclosure may include a solidcarrier capable of adsorbing the nucleic acids containing in a sample ofa body fluid, for example blood (whole blood, plasma, or serum). The kitmay also contain other components for example, reagents, in concentratedor final dilution form, chromatographic materials for the separation ofthe nucleic acids, aqueous solutions (buffers, optionally also inconcentrated form for final adjusting by the user) or chromatographicmaterials for desalting nucleic acids which have been eluted with sodiumchloride.

In embodiments, the present disclosure includes a kit in which materialsmay be included for purifying nucleic acids, for example, inorganicand/or organic carriers and optionally solutions, excipients and/oraccessories. Such agents are known and are commercially available. Forsolid phase nucleic acid isolation methods, many solid supports havebeen used including membrane filters, magnetic beads, metal oxides, andlatex particles.

In addition, in embodiments a kit of the present disclosure can alsocontain excipients such as, for example, a protease such as proteinaseK, or enzymes and other agents for manipulating nucleic acids, e.g., atleast one amplification primer, nucleic acid bases (A, T, G, C, and/orU), and enzymes suitable for amplifying nucleic acids, e.g., DNase, anucleic acid polymerase and/or at least one restriction endonuclease.Alternatively, a commercial polymerase chain reaction kit may be used toamplify the DNA samples.

Method of Detecting Beta Cell Death

Provided herein is a method for detecting circulating beta cell DNA inhuman islet transplant recipients using qMSP starting at one daypost-transplantation and continuing on through several weeks, e.g., twoor more weeks. The DNA may be derived from a biological sample. Forexample, the biological sample may be whole blood, plasma, or serum of ahuman.

In embodiments, a higher persistent signal of unmethylated insulinpromoter DNA in the whole blood compared to the signal from the plasmafraction is achieved. In embodiments of the present disclosure, thepresence of blood cells protects the beta cell DNA in circulation. Inembodiments of the present disclosure, the qMSP assay to human samplesis used to investigate the early stages of human T1D.

In embodiments, the present disclosure includes a method of detectingbeta cell death in a human subject in which detecting unmethylation of aCpG site at −19 bp, −69 bp, −135 bp, −206 bp, and/or −357 bp (upstreambp location from the transcription start site) of the insulin genepromoter detects beta cell death in the human subject. In embodimentsthe method may include that CpG sites at −102, −180, −234 and −345 bp ofthe human insulin gene are unmethylated in stomach, spleen, lung, liver,kidney, colon, breast, blood, and beta cells of the human subject. Inembodiments, the present disclosure may include that the detection ofunmethylation at a CpG site at −102, −180, −234 and −345 bp does notdetect beta cell death and/or early stages of T1D in a subject.

In embodiments, the present disclosure may include a method of detectingeight CpG sites in the exon 2 located at positions +254, +273, +304,+331, +367, +374, +401, and +404 bp; two sites in intron 1 (+127 and+139 bp) and two sites in intron 2 (+456 and +482 bp) relative to thetranscription start site of the human insulin gene. In embodiments, themethod may include that CpG sites at exon 2 are unmethylated in insulingene of blood, breast and liver cells. In embodiments, the method mayinclude that detecting unmethylation in the exon 2 is not indicativebeta cell death. In embodiments the present disclosure may include thatthe detection of unmethylation at a CpG site at exon 2 of the insulingene does not detect beta cell death.

In embodiments the present disclosure may include a method of detectingbeta cell death in a human subject at risk of developing type 1diabetes. In embodiments, the method of the present disclosure includesthe qMSP assay, which is used using blood samples, e.g., whole blood,plasma, or serum, from a human subject. In embodiments, the humansubject is a clinical islet transplant patient. In embodiments, bloodsamples, e.g., whole blood, plasma, or serum, from human subjects, e.g.,islet transplant recipients are obtained prior to transplantation (TX).In embodiments, samples, e.g., whole blood, plasma, or serum, may beobtained from human subjects on 1 or more days after islet transplant.For example, samples may be obtained after 1 day, 2 days, 3 days, 4days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days,13 days, 14 days or more after islet transplant.

Type 1 diabetes (T1D) results from the immune-mediated destruction ofthe insulin-secreting beta cells of the pancreas. The DNA encoding thehuman insulin gene promoter is uniquely unmethylated in beta cells. Inembodiments, the present disclosure includes a methylation-specific PCR(MSP) assay for unmethylated insulin DNA to identify circulating betacell DNA as a measure of beta cell death. In embodiments, the methoddisclosed may include a number of patients, e.g., 10 patients with newonset of T1D age 12 years and older starting within the first 3 monthsof diagnosis, with evaluations throughout their first yearpost-diagnosis focusing on evidence of beta cell loss as well asglycemic control. In embodiments, all T1D patients of the presentdisclosure may be diagnosed based on American Diabetes Association (ADA)criteria of elevated blood glucose and, HbA1c, as well as the presenceof one or more positive autoantibody titers (such as insulin, GAD65 andIA-2 antibodies). In embodiments, blood samples can be collected atdiagnosis, 1, 2, 4, 6, 9, and 12 months post-diagnosis and analyzed byMSP assays. In addition, 90 min stimulated C-peptide level following amixed-meal tolerance tests (MMTT) may be measured at baseline andquarterly to measure of residual beta cell mass. The longitudinalrelationship between these metabolic parameters and the appearance ofbeta cell DNA in circulation may be analyzed using the MSP assay. Inembodiments the present disclosure includes that 60% and 70% of patientsmay be positive for GAD65 and IA-2 autoantibodies, respectively. Inembodiments, HbA1C can range between 9.1% and 18.5% at diagnosis andsubsequently decrease after initiation of insulin therapy. Inembodiments, stimulated C-peptide levels at diagnosis may be very lowbut increases with meal stimulation once the patient started insulintherapy, but may decline again over time. In embodiments, the low levelsof C-peptide at diagnosis can be a reflection of the toxicity ofhyperglycemia to the beta cells and the subsequent improvement withinitiation of insulin therapy and control of hyperglycemia probablyreflected improvement of beta cell function with glucose control. Inembodiments, most of the patients may show a high signal of C-peptideafter stimulation but this may be insufficient to control hyperglycemia,indicating that patients still had residual partial beta cells function.Using the MSP assay, in embodiments the present disclosure includes amethod by which significantly increased relative unmethylation ratio(RUR) of insulin DNA can be observed, which is compatible with the knowntimeline of beta cell death in early onset T1D. In embodiments, adecrease in the RUR is observed following the development of diabetessuggesting the arrest of further beta cell death in the pancreas. Inembodiments the MSP assay of the present disclosure includes thatdestruction of beta cells may occur as a result of episodic attacks ofautoimmunity in T1D. In embodiments, the present disclosure includesthat patients may be in a “honeymoon period” during which the ongoingbeta cell death is delayed. In embodiments, the MSP assay of the presentdisclosure includes an effective method to monitor beta cell destructionin early T1D and is useful in tracking established and innovativemeasures to ameliorate the disease.

In embodiments, the present disclosure includes methods and compositionsto detect tissue-specific methylation pattern in the INS promoter inhuman beta cells, which distinguishes beta cell DNA from DNA of othertissues. In embodiments, the present disclosure includes that not allmethylation of CpG sites in INS promoter of human beta cells aretissue-specific. In embodiments, the present disclosure includes amethod by which methylation of the human INS exon 2 is found to be notdifferentially methylated and, therefore, not suitable for targeting intissue-specific diagnostics.

Method of Treating Autoimmunity Against Insulin Producing Beta Cells

Provided herein is a method for treating an autoimmunity against insulinproducing beta cells in a subject having unmethylated CpG at −19 bp, −69bp, −135 bp, −206 bp, or −357 bp relative to the transcription startsite of an insulin gene promoter, determined by the method as set forthin this disclosure. In embodiments, the subject, e.g., a human subjectis diagnosed with autoimmunity against insulin producing beta cells. Inembodiments, the subject has type 1 diabetes (T1D). In embodiments, thesubject having unmethylated CpG at −19 bp, −69 bp, −135 bp, −206 bp, or−357 bp relative to the transcription start site of an insulin genepromoter is treated for T1D by administering to the subject an activeagent for treating T1D, thereby treating T1D of said subject. Inembodiments, the active agent insulin. In embodiments, the active agentis rapid-acting insulin or long-acting insulin. In embodiments, theinsulin is administered by injection or an insulin pump. In embodiments,the active agent is cyclosporine, anti-CD3 antibody, or anti-CD20antibody. In embodiments, effective treatments is a combination ofagents like anti-CD3 and/or cyclosporine, and further includingantigen-specific therapies such as vaccines and/or factors to stimulatebeta cell growth. In embodiments, the subject is further treated bytransplanting pancreas or islet cells to the subject.

In embodiments, the subject is treated with growth factor and/orcytokine. In embodiments, the subject is treated with one or more of:growth hormone such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor α and β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, β, and γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-16, IL-17, IL-18, IL-22,IL-23, IL-27, IL-35, IL-35; a tumor necrosis factor such as TNF-α orTNF-β; and other polypeptide factors including LIF and kit ligand (KL).

In embodiments, the subject is treated with immunotherapy. For example,in embodiments, the subject is treated with autoantigen-specificregulatory T cell enriched composition. Regulatory T cells (Treg) are asubset of T cells that functionally suppress the proliferation ofeffector T cell populations which are responsible for pathologicalresponses, both in vitro and in vivo. They are phenotypicallycharacterized as CD4+CD25+ T cells which also express the transcriptionfactor Foxp3. Additionally, they have also been shown to express cellsurface markers such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) andglucocorticoid-induced tumor necrosis factor receptor (GITR). CTLA-4 onTregs can downregulate the co-stimulatory molecules CD80 and CD86 onboth murine and human dendritic cells. Tregs can inhibit theco-stimulatory capability of dendritic cells by CD39-mediatedinactivation of extracellular ATP, which is an inducer of dendriticcells activation.

Regulatory T cells can become activated and more suppressive. Forexample, Glycoprotein A repetitions predominant (GARP; also known asleucine-rich repeat protein 32 [LRRC32]) is a marker for activated Tregsthat is not found in conventional CD4+ cells. CD44 is enhanced on thesurface of activated regulatory T cells and these Tregs then showincreased suppressive function. Tregs with a high surface expression ofCD101 are more activated and more suppressive.

In embodiments, the method includes modulating an autoimmune reaction ina subject, the method comprising (a) obtaining a population ofsubject-compatible cells; (b) producing an autoantigen-specific,autoantigen-specific regulatory T cell enriched composition from saidpopulation of cells; and (c) introducing the composition into saidsubject to modulate the autoimmune reaction in the subject. Inembodiments, the population of cells is obtained from a subject havingunmethylated CpG at −19 bp, −69 bp, −135 bp, −206 bp, or −357 bprelative to the transcription start site of an insulin gene promoter,obtained from a donor distinct from said subject, and/or harvested fromperipheral blood of the subjects. The population of cells obtainedincludes autoantigen-specific regulatory T (Treg) cells, and may bederived from any source in which autoantigen-specific Treg cells exist,such as peripheral blood, the thymus, lymph nodes, spleen, and bonemarrow.

In embodiments, the source of Treg cells may be from cadaveric tissue.The population of cells may be obtained from the subject into whom theTreg-enriched composition is subsequently introduced. The subject can beany mammal having unmethylated CpG at −19 bp, −69 bp, −135 bp, −206 bp,or −357 bp relative to the transcription start site of an insulin genepromoter in whom modulation of an autoimmune reaction is desired.Mammals of interest include, but are not limited to: rodents, e.g. mice,rats; livestock, e.g. pigs, horses, cows, etc., pets, e.g. dogs, cats;and primates, e.g. humans. In embodiments, the subject is an animalmodel of an autoimmune disease. There are numerous, established animalmodels for using T cell epitopes of autoantigens to induce tolerance,including multiple sclerosis (EAE: experimental autoimmuneencephalomyelitis), myasthenia gravis (EMG: experimental myastheniagravis), neuritis (EAN: experimental autoimmune neuritis), and type 1diabetes. In an alternate embodiment, the population of cells isobtained from a donor distinct from the subject. The donor is syngeneic,but can also be allogeneic, or even xenogeneic provided the cellsobtained are subject-compatible in that they can be introduced into thesubject, optionally in conjunction with an immunosuppressive therapy,without resulting in extensive chronic graft versus host disease (GvHD).Allogeneic donor cells are human-leukocyte-antigen (HLA)-compatible, andare typically administered in conjunction with immunosuppressivetherapy. To be rendered subject-compatible, xenogenic cells may besubject to gamma irradiation or PEN110 treatment (Fast, L D et al,Transfusion. February 2004; 44(2):282-5). An autoantigen-specificregulatory T (Treg) cell enriched composition is one in which thepercentage of autoantigen-specific Treg cells is higher than thepercentage of autoantigen-specific Treg cells in the originally obtainedpopulation of cells. In embodiments, at least 75%, 85%, 90%, 95%, or 98%of the cells of the composition are autoantigen-specific regulatory Tcells.

In embodiments, the composition may further include one or moreadditional agents, e.g., a costimulatory agent of Treg, a secondregulatory T cell stimulatory agent, or agents that generally promotethe survival and/or growth of T cells. In embodiments, the costimulatoryagent is an antibody or ligand specific for a TCR costimulator, such asCD28 or GITR, as described below. In embodiments, the costimulatoryagent is an agonist antibody, such as an agonist antibody which binds toCD28. The stimulatory composition for Treg alternatively includes asecond regulatory T cell stimulatory agent. Exemplary stimulatory agentsinclude granulocyte colony stimulating factor, interleukins such asIL-2, IL-6, IL-7, IL-13, and IL-15, and hepatocyte growth factor (HGF).In embodiments, the second stimulating agent is a cytokine, such as aninterleukin, such as interleukin-2.

The term “rapid-acting insulin” and the like refer, in the usual andcustomary sense, to insulin for administration which is formulated toachieve activity in a subject relatively rapidly, e.g., 10, 15, 20minutes, which peaks in activity relatively rapidly, e.g., 15, 30, 45,60 minutes. Rapid-acting insulin can continue to provide activity forseveral hours, e.g., 1, 2, 3, 4, 5, 6 hrs. Exemplary rapid-actinginsulin formulation includes insulin glulisine, insulin lispro, andinsulin aspart, as known in the art.

The term “long-acting insulin” and the like refer, in the usual andcustomary sense, to formulations of insulin which reach the bloodstreamseveral hours after administration, e.g., 1, 2, 3, 4 hours or evenlonger. Long acting insulin tends to regulate (i.e., lower) bloodglucose levels fairly evenly over a 24-hr period. Exemplary long-actinginsulin preparations include insulin detemir and insulin glargine, asknown in the art.

The term “anti-CD3 antibody” and the like refer, in the usual andcustomary sense, to antibodies directed against a component of the CD3(i.e, cluster of differentiation 3) T-cell co-receptor, which assemblyincludes a protein complex composed of four distinct chains in mammals:a CD3γ chain, a CD3δ chain, and two CD3ε chains, as known in the art.Methods for generation of antibodies to CD3 are well known in the art.

The term “anti-CD20 antibody” and the like refer, in the usual andcustomary sense, to antibodies directed against the B-lymphocyte antigenCD20. CD20 is a glycosylated phosphoprotein expressed on the surface ofall B-cells, as known in the art. Methods for generation of antibodiesto CD20 are well known in the art.

System for Detecting Unmethylated DNA

Provided herein is a system for detecting unmethylated DNA. FIG. 8 showsa system diagram illustrating a system 800 for detecting unmethylatedDNA in a subject, in accordance with some example embodiments. Referringto FIG. 8, in some example embodiments, the system 800 may be realizedin digital electronic circuitry, integrated circuitry, speciallydesigned application specific integrated circuits (ASICs), fieldprogrammable gate arrays (FPGAs) computer hardware, firmware, software,and/or combinations thereof. The system 800 may be configured tocommunicate with one or more devices (e.g., personal computers,workstations, tablet personal computers, and/or smartphones) via a wiredand/or wireless network 820. For example, as shown, the system 800 maycommunicate with a device 830.

In some example embodiments, the system 800 may include one or moreprocessor to implement a plurality of modules including, but not limitedto, a DNA data collection module 210, a detection module 212, adiagnostic module 214, a treatment module 216, and a user interfacemodule 218. The system 800 may include additional and/or differentmodules without departing from the scope of the present disclosure.

The DNA data collection module 210 may be configured to collect DNAdata. In some example embodiments, the DNA data collection module 210may collect DNA data that has been generated by isolating DNA from asample (e.g., fluid, tissue) of a subject. To generate the DNA data, theisolated DNA may be further subject to modification and/oramplification. For example, modifying the isolated DNA may includemodifying unmethylated cystosine in the DNA to produce DNA that includesuridine. Thereafter, the DNA including uridine may be amplified throughpolymerase chain reaction (PCR) (e.g., qMSP) with methylation specificprimers.

According to some example embodiments, the DNA data may includesequencing data that corresponds to the amplified DNA. As such, in someembodiments, the DNA data collection module 210 may be communicativelycoupled to a DNA sequencer (not shown) adapted to automate thesequencing of DNA that has been isolated, modified, and amplified asdiscussed above. In addition, in some example embodiments, the DNA datacollection module 210 may be communicatively coupled with one or moreDNA processing apparatuses (not shown) via the network 820. For example,the DNA data collection module 210 may be coupled with one or moreapparatuses adapted to automate the performance DNA isolation (e.g.,robotic fluid handling systems), modification, and amplification (e.g.,thermal cycler).

The detection module 812 may be configured to detect unmethylated DNAbased on DNA data (e.g., collected by the DNA data collection module810). For example, in some example embodiments, the detection module 812may be configured to detect unmethylated promoter DNA at a CpG site ofthe insulin gene. The detection of unmethylated insulin gene promotermay be used as a measure of beta cell death and a prognostic indicatorfor autoimmunity resulting in T1D.

The diagnostic module 814 may be configured to provide a diagnosisand/or prognosis for a subject based on results from the detectionmodule 812. In example embodiments, based on the unmethylated DNAdetected in a subject, the diagnostic module 814 may provide a prognosisof whether the subject is at risk for developing T1D. For example, thediagnostic module 814 may perform statistical analysis (e.g., using QUMAand Fisher exact test) on the detected unmethylated DNA to generate aprognosis for a subject.

Alternatively or additionally, based on the detected unmethylated DNA inthe subject, the diagnostic module 814 may also be able to provide adiagnosis of the presence of actual ongoing T1D in the subject. Thediagnostic module 814 may be able to provide a diagnosis of the presenceof actual ongoing T1D before the subject exhibits any loss of metaboliccontrol.

The treatment module 816 may be configured to devise a treatment planfor a subject based on results from the diagnostic module 814. In someexample embodiments, the treatment module 816 may determine, based on asubject's diagnosis and/or prognosis, a treatment plan that includes,but is not limited to, preventative care, medication, and/or islet celltransplantation.

The user interface module 818 may be configured to generate a userinterface through which a user (e.g., a physician) may interact with thesystem 800. For example, the user interface module 818 may provide oneor more user interfaces configured to provide a prognosis and/ordiagnosis from the diagnostic module 814 and/or a treatment plan fromthe treatment module 816. The user interfaces may be graphic userinterfaces (GUIs) adapted to provide visual outputs to and/or receiveinputs from the user.

FIG. 9 shows a flowchart illustrating a process 900 for detectingunmethylated DNA in a subject, in accordance with some exampleembodiments. Referring to FIGS. 8-9, the process 900 may be performed bythe system 800.

At 902, the system 800 (e.g., the DNA data collection module 810) maycollect DNA data associated with a subject. For example, in some exampleembodiments, the system 800 (e.g., the DNA data collection module 810)may collect DNA data that has been generated by isolating a subject'sDNA from a sample (e.g., fluid, tissue). The isolated DNA may have beenfurther modified and/or amplified. According to some exampleembodiments, the DNA data may be sequencing data (e.g., from a DNAsequencer) that corresponds to the amplified DNA.

At 904, the system 800 (e.g., the detection module 812) may detectunmethylated DNA at a CpG site in an insulin gene promoter based on theDNA data. In some example embodiments, the detection of unmethylatedinsulin gene promoter may be used as a measure of beta cell death and aprognostic indicator for autoimmunity resulting in T1D.

At 906, the system 800 (e.g., the diagnostic module 814) may provide aprognosis and/or a diagnosis for the subject based on detectedunmethylated DNA. In some example embodiments, based on unmethylated DNAdetected in a subject, the system 800 may provide a prognosis of thesubject's risk for developing T1D and/or a diagnosis of actual ongoingT1D (e.g., before subject exhibits loss of metabolic control).

At 908, the system 800 (e.g., the treatment module 816) may provide atreatment plan based on the prognosis and/or diagnosis. For example, insome example embodiments, the system 800 may determine a treatment planthat includes, but is not limited to preventative care, medication,and/or islet cell transplantation.

The process 900 can include additional and/or different operationswithout departing from the scope of the present disclosure. One or moreoperations of the process 900 may be omitted and/or repeated withoutdeparting from the scope of the present disclosure.

One or more aspects or features of the subject matter described hereincan be realized in digital electronic circuitry, integrated circuitry,specially designed application specific integrated circuits (ASICs),field programmable gate arrays (FPGAs) computer hardware, firmware,software, and/or combinations thereof. These various aspects or featurescan include implementation in one or more computer programs that areexecutable and/or interpretable on a programmable system including atleast one programmable processor, which can be special or generalpurpose, coupled to receive data and instructions from, and to transmitdata and instructions to, a storage system, at least one input device,and at least one output device. The programmable system or computingsystem may include clients and servers. A client and server aregenerally remote from each other and typically interact through acommunication network. The relationship of client and server arises byvirtue of computer programs running on the respective computers andhaving a client-server relationship to each other.

These computer programs, which can also be referred to as programs,software, software applications, applications, components, or code,include machine instructions for a programmable processor, and can beimplemented in a high-level procedural and/or object-orientedprogramming language, and/or in assembly/machine language. As usedherein, the term “machine-readable medium” refers to any computerprogram product, apparatus and/or device, such as for example magneticdiscs, optical disks, memory, and Programmable Logic Devices (PLDs),used to provide machine instructions and/or data to a programmableprocessor, including a machine-readable medium that receives machineinstructions as a machine-readable signal. The term “machine-readablesignal” refers to any signal used to provide machine instructions and/ordata to a programmable processor. The machine-readable medium can storesuch machine instructions non-transitorily, such as for example as woulda non-transient solid-state memory or a magnetic hard drive or anyequivalent storage medium. The machine-readable medium can alternativelyor additionally store such machine instructions in a transient manner,such as for example, as would a processor cache or other random accessmemory associated with one or more physical processor cores.

To provide for interaction with a user, one or more aspects or featuresof the subject matter described herein can be implemented on a computerhaving a display device, such as for example a cathode ray tube (CRT) ora liquid crystal display (LCD) or a light emitting diode (LED) monitorfor displaying information to the user and a keyboard and a pointingdevice, such as for example a mouse or a trackball, by which the usermay provide input to the computer. Other kinds of devices can be used toprovide for interaction with a user as well. For example, feedbackprovided to the user can be any form of sensory feedback, such as forexample visual feedback, auditory feedback, or tactile feedback; andinput from the user may be received in any form, including, but notlimited to, acoustic, speech, or tactile input. Other possible inputdevices include, but are not limited to, touch screens or othertouch-sensitive devices such as single or multi-point resistive orcapacitive track pads, voice recognition hardware and software, opticalscanners, optical pointers, digital image capture devices and associatedinterpretation software, and the like.

The subject matter described herein can be embodied in systems,apparatus, methods, and/or articles depending on the desiredconfiguration. The implementations set forth in the foregoingdescription do not represent all implementations consistent with thesubject matter described herein. Instead, they are merely some examplesconsistent with aspects related to the described subject matter.Although a few variations have been described in detail above, othermodifications or additions are possible. In particular, further featuresand/or variations can be provided in addition to those set forth herein.For example, the implementations described above can be directed tovarious combinations and subcombinations of the disclosed featuresand/or combinations and subcombinations of several further featuresdisclosed above. In addition, the logic flows depicted in theaccompanying figures and/or described herein do not necessarily requirethe particular order shown, or sequential order, to achieve desirableresults.

The following examples are provided as illustrations of variousembodiments of the disclosure but are not meant to limit the disclosurein any manner.

EXAMPLES Example 1: Evaluation of qMSP for Detection of Circulating BetaCell-Specific DNA in an Autoimmune Mouse Model

MSP Detects Beta Cell Death at the Onset of Insulitis in Non-ObeseDiabetic (NOD) Autoimmune Mouse Model

As part of this study, development of diabetes in the NOD autoimmunemouse model which shares many similarities with type 1 diabetes (T1D) inhumans was monitored and assessed by qMSP assays which determineautoimmune-mediate loss of beta cells. The mice (measured every twoweeks, n=5) became significantly hyperglycemic (>200 mg glucose/dL) atweeks 16 and 18 (p=0.01 and p=0.004, respectively) (FIG. 1A). The dashedline in FIG. 1A at 200 mg/dL indicates the hyperglycemic threshold.However, the lymphocyte infiltration of the islets (insulitis) wasmildly present even at 8 weeks and showed a significant increase by weekand remained high through weeks 12, 14, 16 and 18 (p=0.0007, p=0.01,p=0.009, p=0.001, and p=0.0007, respectively compared with week 8 (FIG.1B). As depicted in FIG. 1B, the degree of insulitis scored was noinsulitis (white), peri-insulitis (dotted), mild insulitis (hatched) andinvasive insulitis (black) for pancreatic sections of the indicatedgroups (n=5) stained with H&E. Concomitantly, there was a significantrise in circulating unmethylated beta cell-specific DNA starting at week10 (FIG. 1C) which remained elevated at weeks 12, 14, and 16 (p<0.0001,p=0.0053, p=0.0008, and 0.04, respectively compared with week 8) untilit dropped to baseline levels at week 18. In a spontaneous diabeticmodel, MSP methodology is capable of detecting beta cell death at theonset of insulitis and six weeks prior to the rise in blood sugar.

Example 2: Methylation Pattern of the Human Insulin Gene (INS) Promotor

Isolation of Genomic DNA

Genomic DNA was obtained from human tissues using NucleoSpin Tissue(Clontech, Mountain View, Calif.). In case of plasma obtained from bloodof transplant recipient, gDNA was purified using QIAamp MinElute VirusSpin Kit and gDNA obtained from blood of transplant recipient waspurified using QIAamp Blood Medi Kit (QIAGEN, Valencia, Calif.).

PCR Cloning of a Fragment from Insulin Gene

Primers H-INS-pro-For and H-INS-exon2-Rev (Table 3) were used to amplifya 900 bp fragment from human gDNA containing the promoter, intron 1,exon 2, and intron 2 of the human insulin gene (INS). The PCR productwas cloned into pCR2.1-TOPO plasmid vector (Invitrogen, Carlsbad,Calif.) and used for development of the assay. The cloned sequence wasconfirmed by the DNA Sequencing/Solexa Core at the Beckman ResearchInstitute of City of Hope using M13F and M13R primers.

Bisulfite Genomic Sequencing

Nucleotide sequence of the human INS (GeneID: 3630) gene was obtainedfrom Genbank and the potential methylation sites (i.e. CG dinucleotides)were identified. Genomic DNA was isolated from various tissues asdescribed above and were treated with EZ DNA methylation-gold kit (ZymoResearch, Orange, Calif.) according to the manufacturer'srecommendation. The human INS gene was then amplified with pairs ofgene-specific primers for promoter and exon 2 (Table 3) in a mixturecontaining 100 ng bisulfite modified DNA as a template and DNA HotStar-Taq polymerase (QIAGEN, Valencia, Calif.). Each PCR fragment was TAcloned into pCR2.1-TOPO vector and sequenced as described above. Eachpattern resulted from 18-59 clones of INS promoter or 5-33 clones of INSexon 2 obtained from 6 different individuals for blood tissue and 3individuals from other tissues as indicated in (Table 4). Statistics ofeach CpG site were done using the QUMA computer program which performs aFisher exact test.

TABLE 3 Oligonucleotides used in this study. Designation SequencePrimers for methylation mapping of human INS promoter 1 HINSex2-For5'-GGTTTAGGATTTTAGGGTGGTT-3' (SEQ ID NO: 7) 2 HINSex2-Rev5'-CCCCCTTCTACCCATACTAAAT-3' (SEQ ID NO: 8)Primers for methylation mapping of human INS exon 2 1 HuINS420-For5'-TGTGGGGATAGGGGTTTGGGGATAGT A-3' (SEQ ID NO: 9) 2 HuINS420-Rev5'-CCTCTTCTAATACAACCTATCCTAAA AAACTAAAAACTAC-3' (SEQ ID NO: 10)Primers for cloning human INS gene 1 H-INS-pro-For5'-TGTGGGGACAGGGGTCTGGGGAC A-3' (SEQ ID NO: 11) 2 H-INS-exon2-Rev5'-AGCCTCCTGCCCCCTTCTGCCCA T-3' (SEQ ID NO: 12) Primers for qMSP P20H-Pro-Bisulf-For1 5'-ATAGGGGTGTGGGGATAGGGGTTTGGGGATAGTAGT-3' (SEQ ID NO: 1) P21 H-Pro-Bisulf-Rev15'- AACCCATCTCCCCTACCTCTCAACC CCTACCA-3' (SEQ ID NO: 2) P38H-Pro-BS-For4 5'-TGGGTTTTTGGTTAAGATTTTAATGA TTT-3' (SEQ ID NO: 3) P39H-Pro-BS-Rev5 5'-CAACAAATAACTAAAAACTAAAACTA CAATTTCCA-3' (SEQ ID NO: 4)Primers for a BSP P40 MSP-For1 5'-ATAGGGGTGTGGGGATAGGGGTTTGGGGATAGTA-3' (SEQ ID NO: 5) P41 MSP-Rev1 5'-CAAAACCCATCTCCCCTACCTCTCAACCCCTAC-3' (SEQ ID NO: 6)

TABLE 4 Mapping of human insulin promoter and exon 2 regions. PromoterExon 2 Organ Number of donors Number of Number of Number of tissuestissue clones donors tissue clones Beta cells 3 26 2 12 Blood 6 59 5 25Breast 3 22 2 5 Colon 3 27 3 22 Kidney 3 22 3 14 Liver 3 20 3 33 Lung 324 3 19 Spleen 3 21 3 21 Stomach 3 18 3 13INS Promoter in Human Beta Cells Exhibited a Tissue-Specific MethylationPattern to Distinguish Beta Cell DNA from DNA of Other Tissues

The promoter of the human INS contains nine potential methylation (CpGdinucleotide) sites that are located at positions −357, −345, −234,−206, −180, −135, −102, −69 and −19 bp relative to the transcriptionstart site (TSS), as indicated in FIG. 2. A broader examination of thetissue-dependent methylation of the human insulin gene was performed.

Genomic DNA from nine different tissues including an enriched beta cellfraction were subjected to bisulfite sequencing of the INS gene to maptheir respective CpG methylation patterns (FIG. 2). Six different donorsfor blood and 3 donors for other tissues were used for each sample(human blood, breast, colon, kidney, liver, lung, spleen, stomach andhuman beta cells ‘Islet cell fraction’≈70% islet cells). FIG. 2 displaysthe position and the percentage of unmethylation (white bars) tomethylation (black bars) for each CpG. Analysis of the bisulfitesequencing data revealed that most of the CpG sites in the INS promoterwere uniquely unmethylated in pancreatic beta cells but predominantlymethylated in other tissues (FIG. 2). Not all of the sites exhibited thesame degree of tissue-dependent methylation. The CpGs at −102, −180,−234, and −345 bp were substantially unmethylated in all the tissuesexamined and were not statistically different from beta cells (FIG. 2).Conversely, sites −19, −69, −135, −206, and −357 bp relative to TSS wereunmethylated in beta cells but not in other tissues, and displayed asignificant tissue-specificity. Only certain sites in the human INSpromoter exhibited a tissue-specific DNA methylation pattern andtherefore only these sites can differentiate between beta cells andother tissues.

Example 3: Methylation Pattern of the Human Insulin Gene Exon 2, Intron1 and Intron 2

Exon 2 and the surrounding regions were bisulfite sequenced to determinewhether other regions of the human INS were also preferentiallyunmethylated. FIG. 3 depicts a histogram of the tissue methylationpattern of the human INS exon 2 (8CpG), intron 1 (2 CpG) and intron 2 (2CpG), and the positions of the twelve CpG sites relative to the TSS areindicated in FIG. 3. The bars display the position and percentage ofunmethylation (white bars) to methylation (black bars) for each CpG.Each pattern resulted from 5 to 33 clones obtained from 5 individual'sblood, 2 individual's beta cells and breast, and 3 individuals for theother tissues. FIG. 3 shows that there were eight CpG sites in exon 2located at positions +254, +273, +304, +331, +367, +374, +401, and +404bp relative to the TSS. Two other sites in intron 1 (+127 and +139) andtwo sites in intron 2 (+456 and +482) were also examined. As in thepromoter, these sites were mostly unmethylated in beta cells andmethylated in colon, kidney, lung, spleen and stomach (FIG. 3). However,exon 2 was also found to be predominantly unmethylated in blood, breastand liver cells. Therefore, the human INS exon 2 region was notappropriate for targeting beta cell DNA in circulation in the qMSP assaysince it did not exhibit a beta cell-specific pattern, especially inblood, the major background signal of the assay.

Example 4: Dose Dependent Amplification of Methyl Specific andBisulfate-Specific PCR Primer Sets

Nested Methylation-Specific PCR (MSP)

Quantitative PCR was performed with a 7500 Real-time PCR instrument(Applied Biosystems, Foster City, Calif.). In First-Step PCR, eachreaction contained 20-30 ng of bisulfite-treated DNA, 12.5 μl QuantiTectSYBR® Green PCR (QIAGEN, Valencia, Calif.) and 500 nM each forward andreverse primer (Table 3) in a total volume of 250. Thermal cycling wasinitiated with an enzyme activation step of 15 min at 95° C., followedby 15 cycles of 95° C. for 15 s, 60° C. for 30 s, and, 72° C. for 30 s.The PCR products were purified using QIAquick PCR Purification Kit(QIAGEN, Valencia, Calif.). In Second-Step PCR, the products from thefirst reaction were used as template for a qPCR with nested primers(Table 3). The reactions were initiated with for 15 min at 95° C.,followed by 40 cycles of 95° C. for 15 s, 57° C. for 30 s, and, 72° C.for 30 s. The quantification cycle (Cq) was determined for each reactionwith methylation-specific primers (MSP) and bisulfite-specific primers(BSP) and the ratio of unmethylated to total amplifiablebisulfite-treated DNA was calculated using the Relative UnmethylationRatio (RUR) as previously described by Husseiny et al. as RelativeExpression Ratio (RER). The second-step reaction Cq values were between15 and 40. Negative controls without DNA did not yield products in thefirst-step reaction.

Human Subjects

Human tissues and islet cells were isolated. To enrich beta cells,islets were dissociated with TrypLE (Invitrogen, Carlsbad, Calif.) andstained with Newport Green (Invitrogen, Carlsbad, Calif.) beforeenriching by fluorescent-activated cell sorting. Normal human tissuessuch as liver, breast, colon, kidney, lung, spleen, and stomach wereobtained from the Pathology Core at City of Hope. Blood samples werecollected from normal healthy controls and from patients before andafter islet transplantation.

Quantitative Methylation-Specific PCR

The human assay involved six differentially methylated sites. A nestedPCR technique was developed and used, which interrogated additionalmethylation sites while improving both the specificity and sensitivityof the assay. Primers were designed to recognize only unmethylated DNAas found in beta cells (FIG. 4A). Primers P20 and P21 aremethylation-specific primers that amplify unmethylated DNA only (FIG.4A, dashed arrows) targeting CpGs at −357 and −69 and together produce aproduct of 350 bp (top agarose gel depicted in FIG. 4B). Primers P38 andP39 target CpGs −206 and −135, respectively, and produce a product of130 bp (bottom agarose gel in FIG. 4B). In addition, two primers, P40and P41, target the regions just upstream and downstream of P20 and P21,respectively, and were not aligned with any CpG site. P40 and P41primers are bisulfate-specific primers (BSPs) that amplify bothmethylated and unmethylated DNA and are shown in FIG. 4A as solidarrows. The P40 and P41 primers amplify a 350 bp product from bothmethylated and unmethylated DNA (middle agarose gel depicted in FIG.4B), and therefore provide a measure of total amplifiable insulin genepromoter sequences. These primer sets were evaluated using serialdilutions of the cloned unmethylated insulin gene as a template. Asshown, each MSP and BSP primer set exhibited dose dependentamplification ranging from 10⁶ copies to as few as 5 copies of theunmethylated sequences (FIG. 4B).

Example 5: Quantitative Analysis of Bisulfate-Specific (BSP) and qMSPAssays

Statistical Analysis

Statistical significance between samples was tested with a two-tailedStudent's t-test for unpaired values or two-way analysis of variance(ANOVA) between human islets and other tissues (colon and blood) usingGraphPad Prism 6 software. Statistical significance was defined as aP-value of <0.05, <0.01, and <0.001. Statistical analysis of DNAmethylation was done using QUMA (http://quma.cdb.riken.jp/) whichperforms a Fisher exact test on the methylation status of individual CpGsites using P<0.1, and P<0.01. Data are expressed as mean±SEM.

Qualitative Analysis of Standard Curves of BSP and qMSP Assays

Quantitative analysis of the standard curves shows that the BSP and qMSPassays were linear over a 10⁵-fold range of template concentrations(FIG. 4C). FIG. 4C depicts graph of real time SYBR® Green PCR datashowing linearity of Cq versus log copy number of unmethylated plasmidfrom 5 to 10⁶ copies. For nested PCR, the two MSP assays were appliedsequentially, i.e. amplification with P20/P21 followed by P38/P39.Variation across the nested MSP curve ranged from 2.83% to 6.58% (Table1). Furthermore, the standard curve parameters (Table 2) were highlyreproducible for both nested qMSP (efficiency=85.19%±1.37 SD,slope=−3.737±0.05 SD, R²=0.979±0.004 SD; n=5 experiments) and qBSP(efficiency=73.44%±7.81 SD, slope=−4.212±0.32 SD, R²=0.989±0.01 SD; n=5experiments).

Example 6: Assay Specificity Increases with Increasing CpG SitesInterrogated in the Assay

Primers sets (from Example 4, FIG. 4A) were tested for specificity andsensitivity employing serially diluted bisulfite converted gDNA fromhuman islets, blood, and colon as templates for qMSP. Fold changes inunmethylation were calculated by the Relative Unmethylation Ratio (RUR)for each sample (See FIG. 1C for RUR description) in which the level ofbeta cell DNA (qMSP) was normalized for total amplifiable sequences(qBSP). To assess the effect of targeting 2 versus 4 CpG sites, thenested reaction using P38/P39 was preceded by a first reaction usingeither BSP primers (P40/P41) to target a total of 2 sites or MSP primers(P20/P21) to target 4 sites. The assay interrogating 2 CpG sitesexhibited a highly significant specificity for islets over blood andcolon (FIG. 5A). However, the assay targeting 4 sites showed a greaterdifference in signal between islets and other tissues, indicating thatthe specificity of the assay was increased by increasing the number ofCpG sites interrogated in the assay (FIG. 5B).

Example 7: Quantitative MSP for Monitoring Circulating Beta Cell DNA inIslet Transplant Patients

The application of the qMSP assay to human studies was assessed usingblood samples from clinical islet transplant patients. Samples wereobtained from islet recipients (n=6) prior to transplantation (TX) andon post-transplant days 1 and 14. These were compared with blood (FIG.6A) from healthy donors (n=6) using the qMSP assay. Plasma fractions(FIG. 6B) were also prepared and compared with the results of wholeblood to determine whether plasma was a better starting point for theassay. Prior to transplantation, there was no significant difference inthe qMSP signal between the patient samples and normal controls (FIG.6A; Mann-Whitney U test; p=0.92). However, the qMSP signal rosesignificantly the day after islet transplantation (Wilcoxon test;p=0.005) and remained elevated for at least fourteen days (Wilcoxontest; p=0.004) in the whole blood samples (FIG. 6A). In plasma samples,the signal also rose significantly on day 1 (FIG. 6B; Wilcoxon test;p=0.003), though in contrast to whole blood, fell again by day 14(Wilcoxon test; p=0.58). Beta cell DNA is associated with cells in theblood and prolongs the qMSP signal. qMSP can be used herein to monitorbeta cell DNA in human clinical samples and the duration of the signalis longer in whole blood than in plasma.

Example 8: MSP Assay and Mixed Meal Tolerance Test on New Onset T1DPatients

Patient Characterization

Patients in the study were newly diagnosed T1D patients, 12 years orolder, healthy individuals with no cancer and not pregnant. Patientswere also willing to comply with the scheduled visits to the clinicunder the continuing care of their endocrinologists. The patients werediagnosed on American Diabetic Association (ADA) criteria of elevatedblood glucose and, HbA1c, as well as the presence of one or morepositive antibody titers.

Study Design and Methods

Fasting blood samples were drawn at time of diagnosis, 2 weeks, 1, 2, 4,6, 9, and 12 months. Blood samples were analyzed with fasting bloodglucose levels, fasting serum c-peptide, HbA1c, serum auto-antibodytiers and MSP assays. To evaluate residual islet mass, stimulatedc-peptide level following a mixed-meal tolerance test (MMTT) at baselineand at 3 month intervals was measured. The longitudinal relationshipbetween the metabolic parameters and the appearance of beta cell DNA incirculation was analyzed by MSP.

TABLE 5 Patient Characteristics* GAD65 Islet Antigen Ab Insulin Ab 2(IA-2) Gender (nmol/L) (nmol/L) (nmol/L) HbA1C # (M/F) (≤0.02) (0.0 ·0.02) (≤0.02) (4.8%-5.9%) Age/Yr 1 M 0.01 0.01 11.5 12.70% 15 2 F 0 03.23 18.50% 14.3 3 M 0.18 0 0 12.90% 16.5 4 M 0.17 0 2.99  9.10% 14.7 5M 5.91 0 1.97 11.60% 12.8 6 M 0.06 0 0.14 12.80% 14.3 7 M 0.08 0 2.4213.00% 13.3 8 M n/d n/d n/d  7.80% 17 9 M 1.21 0 0.03  7.40% 14.5 10 F 00 0 12.20% n/d *60% are positive for GAD65 Ab, 70% are positive for IA-2Ab, 50% are positive for GAD65 and IA-2b Abs.

Patients were monitored with new onset of T1D age 12 years and olderstarting within the first 3 months of diagnosis. Evaluations were madethroughout the first year post-diagnosis, focusing on evidence of betacell loss as well as glycemic control. All T1D patients were diagnosedbased on ADA criteria of elevated blood glucose and, HbA1c, as well asthe presence of one or more positive autoantibody titers (insulin, GAD65and IA-2 antibodies). Blood samples were collected at diagnosis, 1, 2,4, 6, 9 and 12 months post-diagnosis and analyzed by MSP assay (FIGS.7A-7AA). Results indicated that 60% and 70% of patients were positivefor GAD65 and IA-2 autoantibodies, respectively. HbA1c ranged between9.1% and 18.5% at diagnosis and subsequently decreased after initiationof insulin therapy. Stimulated C-peptide levels at diagnosis were verylow, but increased with meal stimulation once the patient startedinsulin therapy, but declined again over time (FIGS. 7A-7AA, middlehistogram of each patient). At diagnosis, high insulin amounts wererequired and decreased once the patient started insulin therapy. Most ofthe patients showed high signal of C-peptide after stimulation. Usingthe MSP assay, a significantly increased relative unmethylation ratio(RUR) of insulin DNA that is compatible with the known timeline of betacell death in early onset T1D was observed (FIGS. 7A-7AA, left histogramof each patient).

Example 9

A system for detecting unmethylated DNA is provided in this example.FIG. 8 shows a system diagram illustrating a system 800 for detectingunmethylated DNA in a subject, in accordance with some exampleembodiments. Referring to FIG. 8, in some example embodiments, thesystem 800 is realized in digital electronic circuitry, integratedcircuitry, specially designed application specific integrated circuits(ASICs), field programmable gate arrays (FPGAs) computer hardware,firmware, software, and/or combinations thereof. The system 800 isconfigured to communicate with one or more devices (e.g., personalcomputers, workstations, tablet personal computers, and/or smartphones)via a wired and/or wireless network 820. For example, as shown, thesystem 800 communicates with a device 830.

In some example embodiments, the system 800 includes one or moreprocessor to implement a plurality of modules including, but not limitedto, a DNA data collection module 210, a detection module 212, adiagnostic module 214, a treatment module 216, and a user interfacemodule 218. The system 800 includes additional and/or different moduleswithout departing from the scope of the present disclosure.

The DNA data collection module 210 is configured to collect DNA data. Insome example embodiments, the DNA data collection module 210 collectsDNA data that has been generated by isolating DNA from a sample (e.g.,fluid, tissue) of a subject. To generate the DNA data, the isolated DNAis further subject to modification and/or amplification. For example,modifying the isolated DNA includes modifying unmethylated cytosine inthe DNA to produce DNA that includes uridine. Thereafter, the DNAincluding uridine is amplified through polymerase chain reaction (PCR)(e.g., qMSP) with methylation specific primers.

According to some example embodiments, the DNA data includes sequencingdata that corresponds to the amplified DNA. As such, in someembodiments, the DNA data collection module 210 is communicativelycoupled to a DNA sequencer (not shown) adapted to automate thesequencing of DNA that has been isolated, modified, and amplified asdiscussed above. In addition, in some example embodiments, the DNA datacollection module 210 is communicatively coupled with one or more DNAprocessing apparatuses (not shown) via the network 820. For example, theDNA data collection module 210 is coupled with one or more apparatusesadapted to automate the performance DNA isolation (e.g., robotic fluidhandling systems), modification, and amplification (e.g., thermalcycler).

The detection module 812 is configured to detect unmethylated DNA basedon DNA data (e.g., collected by the DNA data collection module 810). Forexample, in some example embodiments, the detection module 812 isconfigured to detect unmethylated promoter DNA at a CpG site of theinsulin gene. The detection of unmethylated insulin gene promoter isused as a measure of beta cell death and a prognostic indicator forautoimmunity resulting in T1D.

The diagnostic module 814 is configured to provide a diagnosis and/orprognosis for a subject based on results from the detection module 812.In example embodiments, based on the unmethylated DNA detected in asubject, the diagnostic module 814 provides a prognosis of whether thesubject is at risk for developing T1D. For example, the diagnosticmodule 814 performs statistical analysis (e.g., using QUMA and Fisherexact test) on the detected unmethylated DNA to generate a prognosis fora subject.

Alternatively or additionally, based on the detected unmethylated DNA inthe subject, the diagnostic module 814 also is able to provide adiagnosis of the presence of actual ongoing T1D in the subject. Thediagnostic module 814 is able to provide a diagnosis of the presence ofactual ongoing T1D before the subject exhibits any loss of metaboliccontrol.

The treatment module 816 is configured to devise a treatment plan for asubject based on results from the diagnostic module 814. In some exampleembodiments, the treatment module 816 determines, based on a subject'sdiagnosis and/or prognosis, a treatment plan that includes, but is notlimited to, preventative care, medication, and/or islet celltransplantation.

The user interface module 818 is configured to generate a user interfacethrough which a user (e.g., a physician) interacts with the system 800.For example, the user interface module 818 provides one or more userinterfaces configured to provide a prognosis and/or diagnosis from thediagnostic module 814 and/or a treatment plan from the treatment module816. The user interfaces are graphic user interfaces (GUIs) adapted toprovide visual outputs to and/or receive inputs from the user.

FIG. 9 shows a flowchart illustrating a process 900 for detectingunmethylated DNA in a subject, in accordance with some exampleembodiments. Referring to FIGS. 8-9, the process 900 is performed by thesystem 800.

At 902, the system 800 (e.g., the DNA data collection module 810)collects DNA data associated with a subject. For example, in someexample embodiments, the system 800 (e.g., the DNA data collectionmodule 810) collects DNA data that has been generated by isolating asubject's DNA from a sample (e.g., fluid, tissue). The isolated DNA isfurther modified and/or amplified. According to some exampleembodiments, the DNA data is sequencing data (e.g., from a DNAsequencer) that corresponds to the amplified DNA.

At 904, the system 800 (e.g., the detection module 812) detectsunmethylated DNA at a CpG site in an insulin gene promoter based on theDNA data. In some example embodiments, the detection of unmethylatedinsulin gene promoter is used as a measure of beta cell death and aprognostic indicator for autoimmunity resulting in T1D.

At 906, the system 800 (e.g., the diagnostic module 814) provides aprognosis and/or a diagnosis for the subject based on detectedunmethylated DNA. In some example embodiments, based on unmethylated DNAdetected in a subject, the system 800 provides a prognosis of thesubject's risk for developing T1D and/or a diagnosis of actual ongoingT1D (e.g., before subject exhibits loss of metabolic control).

At 908, the system 800 (e.g., the treatment module 816) provides atreatment plan based on the prognosis and/or diagnosis. For example, insome example embodiments, the system 800 determines a treatment planthat includes, but is not limited to preventative care, medication,and/or islet cell transplantation.

The process 900 can include additional and/or different operationswithout departing from the scope of the present disclosure. One or moreoperations of the process 900 is omitted and/or repeated withoutdeparting from the scope of the present disclosure.

One or more aspects or features of the subject matter described hereincan be realized in digital electronic circuitry, integrated circuitry,specially designed application specific integrated circuits (ASICs),field programmable gate arrays (FPGAs) computer hardware, firmware,software, and/or combinations thereof. These various aspects or featurescan include implementation in one or more computer programs that areexecutable and/or interpretable on a programmable system including atleast one programmable processor, which can be special or generalpurpose, coupled to receive data and instructions from, and to transmitdata and instructions to, a storage system, at least one input device,and at least one output device. The programmable system or computingsystem may include clients and servers. A client and server aregenerally remote from each other and typically interact through acommunication network. The relationship of client and server arises byvirtue of computer programs running on the respective computers andhaving a client-server relationship to each other.

These computer programs, which can also be referred to as programs,software, software applications, applications, components, or code,include machine instructions for a programmable processor, and can beimplemented in a high-level procedural and/or object-orientedprogramming language, and/or in assembly/machine language. As usedherein, the term “machine-readable medium” refers to any computerprogram product, apparatus and/or device, such as for example magneticdiscs, optical disks, memory, and Programmable Logic Devices (PLDs),used to provide machine instructions and/or data to a programmableprocessor, including a machine-readable medium that receives machineinstructions as a machine-readable signal. The term “machine-readablesignal” refers to any signal used to provide machine instructions and/ordata to a programmable processor. The machine-readable medium can storesuch machine instructions non-transitorily, such as for example as woulda non-transient solid-state memory or a magnetic hard drive or anyequivalent storage medium. The machine-readable medium can alternativelyor additionally store such machine instructions in a transient manner,such as for example, as would a processor cache or other random accessmemory associated with one or more physical processor cores.

To provide for interaction with a user, one or more aspects or featuresof the subject matter described herein can be implemented on a computerhaving a display device, such as for example a cathode ray tube (CRT) ora liquid crystal display (LCD) or a light emitting diode (LED) monitorfor displaying information to the user and a keyboard and a pointingdevice, such as for example a mouse or a trackball, by which the usermay provide input to the computer. Other kinds of devices can be used toprovide for interaction with a user as well. For example, feedbackprovided to the user can be any form of sensory feedback, such as forexample visual feedback, auditory feedback, or tactile feedback; andinput from the user may be received in any form, including, but notlimited to, acoustic, speech, or tactile input. Other possible inputdevices include, but are not limited to, touch screens or othertouch-sensitive devices such as single or multi-point resistive orcapacitive track pads, voice recognition hardware and software, opticalscanners, optical pointers, digital image capture devices and associatedinterpretation software, and the like.

The subject matter described herein can be embodied in systems,apparatus, methods, and/or articles depending on the desiredconfiguration. The implementations set forth in the foregoingdescription do not represent all implementations consistent with thesubject matter described herein. Instead, they are merely some examplesconsistent with aspects related to the described subject matter.Although a few variations have been described in detail above, othermodifications or additions are possible. In particular, further featuresand/or variations can be provided in addition to those set forth herein.For example, the implementations described above can be directed tovarious combinations and subcombinations of the disclosed featuresand/or combinations and subcombinations of several further featuresdisclosed above. In addition, the logic flows depicted in theaccompanying figures and/or described herein do not necessarily requirethe particular order shown, or sequential order, to achieve desirableresults. Other implementations may be within the scope of the followingclaims

What is claimed is:
 1. A nucleic acid comprising SEQ ID NO: 1, 2, 3, or
 4. 2. A nucleic acid complex comprising the nucleic acid of claim 1, wherein said SEQ ID NO: 1, 2, 3, or 4 is contacted to a complementary deoxyribonucleic acid (DNA) sequence to form a hybridized nucleic acid complex, wherein said complementary DNA sequence comprises uridine.
 3. The nucleic acid complex of claim 2, wherein said nucleic acid is hybridized to a CpG site of said complementary DNA sequence.
 4. A deoxyribonucleic acid (DNA) comprising a uridine that is hybridized to a first nucleic acid and a second nucleic acid each independently comprising SEQ ID NO: 1, 2, 3, or 4, to form a hybridized DNA with SEQ ID NO: 1, 2, 3, or 4, wherein said first and said second nucleic acids do not simultaneously comprise the same SEQ ID NO: 1, 2, 3, or
 4. 5. The DNA of claim 4, wherein said first nucleic acid comprises SEQ ID NO: 1 and said second nucleic acid comprises SEQ ID NO:
 2. 6. The DNA of claim 4, wherein said first nucleic acid comprises SEQ ID NO: 3 and said second nucleic acid comprises SEQ ID NO:
 4. 7. The DNA of claim 4, wherein said DNA is derived from a biological sample.
 8. The DNA of claim 7, wherein said biological sample is blood.
 9. The DNA of claim 8, wherein said blood is whole blood, plasma, or serum.
 10. A kit comprising a first nucleic acid and a second nucleic acid each independently comprising SEQ ID NO: 1, 2, 3, 4, 5, or 6, wherein said first and said second nucleic acids do not simultaneously comprise the same SEQ ID NO: 1, 2, 3, 4, 5, or
 6. 11. The kit according to claim 10, wherein said first nucleic acid comprises SEQ ID NO: 1 and said second nucleic acid comprises SEQ ID NO:
 2. 12. The kit according to claim 10, wherein said first nucleic acid comprises SEQ ID NO: 3 and said second nucleic acid comprises SEQ ID NO:
 4. 13. The kit according to claim 10, wherein said first nucleic acid comprises SEQ ID NO: 5 and said second nucleic acid comprises SEQ ID NO:
 6. 14. The kit according to claim 10, further comprising: enzymes, reagents for deamination of cytosine, buffers, vials, plasmid vectors, control DNA, devices for collecting blood and/or tissue samples, or reagents for labeling DNA, or any combinations thereof.
 15. The kit according to claim 14, wherein the enzymes are selected from the group consisting of: thermostable DNA polymerase enzymes, restriction enzymes, and combination thereof. 